By definition a monoclonal antibody is of one Ig subtype, while a polyclonal antibody contains multiple Ig subtypes.  So look at our product isotype description and if it says: IgG1, IgG2a, IgG2b, IgM, etc, these will be monoclonal, whereas: Goat IgG, Rabbit Ig, etc. will be polyclonal. Most of our polyclonal antibodies also have the term poly in the clone name.

Perform a search for your species using our search box at the top of the web page. Alternatively, use our advanced search drop down menu for Species Reactivity and select your choice of species.  For a broad overview of all our cross-reactive antibodies, use our Antibody Cross-reactivity Chart.

On each product data sheet there is a link to all of the available MSDS.  Or check our website under "Support". If your product isn't covered in this section, please contact BioLegend Technical Services.

100% Satisfaction Guarantee. If BioLegend's product does not perform as described on its product data sheet, we'll replace it or refund 100% of the original purchase price

There is an acceptable tolerance range between 2°C and 8°C, for all products recommended to be stored at 4°C.  If your refrigerator at any time has temperatures outside of this range, we recommend you adjust its settings or use a different refrigerator.  

BioLegend does not epitope map antibodies. Sometimes this information is indicated on the datasheets, if published, but generally, we will not know the exact epitope of the antibody.

Most of our products (unless stated otherwise) have a guaranteed shelf life until their expiration date, when under proper storage and handling conditions as instructed on our Product Data Sheets.  For recombinant proteins, the minimum guaranteed shelf-life is 6 months from the date of receipt by the end-user.

For U.S. orders, if products are in-stock and your order was made before the shipping cutoff time (contact cs@biolegend.com for more info), they are shipped out that day for you to receive the next day. We typically do not ship on Fridays as to avoid packages sitting unattended over the weekend. International direct orders are shipped on Mondays and Fridays, and typically arrive within 2 - 10 days, depending on how long they are in customs.

For direct orders shipped outside the U.S., shipping costs typically start at USD $50 for antibody orders and $100 for dry ice shipment. This could vary country to country. Additionally, prepayment is required for direct international shipments. Also, taxes and duties will be paid by the recipient upon shipment delivery. In some countries, the customer needs to apply for an import permit. When ordering from a distributor, the payment terms are more flexible, the customer and technical support is more immediate, and the customer has greater convenience in not having to deal with customs agencies, duties/taxes, and currency exchange.

Most products are shipped at ambient temperature.  Our products are sufficiently stable to maintain optimal performance after overnight shipping.

Check the Product Data Sheets on-line to review applications/characteristics for each clone. If you still have questions, please contact BioLegend Technical Services at:

• Toll Free Phone (U.S., Canada): 1-877-BIOLEGEND (246-5343)
  Phone: 858-455-9588
  Fax: 858-455-9587
• E-mail: Click Here

Antibodies offered per test have been pre-titrated for optimal performance in flow cytometry assays. Antibodies offered per plate have been pre-titrated in ELISA. Cell Biology antibodies offered per µl have been pre-titrated for Western Blot. The antibody packed in an μg format (untitrated) is not necessarily always from the same batch number as a test format (pre-titrated) though the same procedures are performed in the quality control testing. Please also note that not all test formats of the antibodies have lower concentrations than the μg format. It depends on a particular batch tested. If the optimal concentration were determined at 1 μg/test for a particular batch, then it would be 100 μg in the 100 tests. The advantage to using a pre-titrated format is that it provides more convenience for the optimal performance in areas such as flow cytometry assays, especially for beginners who may save time and reagents on titration of the antibody.

Fluorescence to Protein (f:p) ratio tells you the average number of fluorophore molecules that are conjugated to each antibody for any given lot of product. These values can vary widely between manufacturers and even among different lots from the same manufacturer. When using isotype controls, it is important to verify that your control has a similar f:p as that of the primary antibody.

Isotype controls are recommended for every flow experiment. Isotype controls that do not recognize any known proteins will provide you with information on the background staining of an antibody due to its isotype. For multicolor experiments, fluorescence-minus-one (FMO) controls can help you determine the fluorescence spillover from all your other antibodies and can be used to help in gating positive and negative boundaries. Unstained cells can also provide you with a relative measure of the autofluorescence associated with any particular cell type.

Compensation is the process of removing spillover (spectral overlap) from other fluorophores into the detector for your fluorophore of choice. For example, due to the wide emission range of FITC, some of the FITC signal “bleeds” into PE giving you false signal in the PE channel. On newer digital instruments, compensation can be applied automatically using single stain controls.

Fixation with paraformaldyde generally tends to decrease the fluorescence intensity of bound antibodies, particularly with prolonged exposure. This is especially true for nanocrystals and tandem dyes such as those derived from PE and APC. Excessive fixation can also alter the conformation of proteins, causing loss of antibody reactivity to proteins as well.

According to a study (Le Roy C, et al. 2009. Cytometry A. 75:882), APC tandems are degraded through a cell-dependent mechanism.

There are several different methods that can be used to conjugate fluorophores to antibodies. This can depend on the antibody and type of fluorophore being used. One common method is to conjugate the organic fluorophore via primary amines (lysines) on the antibody. Another method conjugates maleimide-labeled fluorophore with antibody via sulfhydryl groups in the hinge region.

We don't perform epitope mapping in-house. We perform our quality control (QC) test with either surface staining or intracellular staining, as indicated in each product datasheet. When surface staining is performed, it means the antibody recognizes an extracellular epitope. In contrast, if the QC is done with intracellular staining, it means that the protein is expressed in this cellular compartment, but it does not necessarily mean that the epitope recognized is exclusively intracellular. The antibody may still be able to detect the protein if it is expressed or exported to the surface of the cell. It is best to further consult the literature related to the particular clone in this matter.

Refer to your instrument manual to determine the specifications of your instrument. You may also be able to find specifications in the software provided with your instrument. Note that many instruments are custom built and may not match the standard manual. Also, many instruments have adjustable filter sets, allowing you to configure your instrument to suitably run many different combinations of fluorophores. Always verify that the instrument you plan to use is capable of detecting the fluorophores in your experimental panel.

Fluorescence is the emission of light by a substance that has absorbed light. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed wavelength. In flow cytometry, fluorophores are used to tag antibodies in order to provide a signal upon detection of an antigen on the sample when exposed to a single wavelength laser.

We have not tested the Maxpar^® Ready antibodies formulated in solution containing EDTA for flow cytometry staining. While it is likely that this will work in majority of the situations, it is best to use the non-EDTA formulated version of the same clone for flow cytometry testing. The presence of EDTA in some situations might negatively affect staining.

The Maxpar^® Ready format antibody clones are formulated in Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and EDTA. The regular purified format clones are formulated in solution that does not contain any EDTA. Both formulations are however without any extra carrier proteins.

Be sure to use the buffers recommended in the protocol that you are using.  BioLegend provides a number of flow cytometry protocols, as well as a page to guide you in selecting the appropriate buffers for your flow cytometry experiments.

Tandem dyes are fluorophores composed of two distinct fluorophores conjugated together. The resulting tandem uses the excitation property of the donor fluorophore and emission property of the acceptor fluorophore, based on the principles of fluorescence resonance energy transfer (FRET). This allows for novel fluorophores with high stokes shifts (separation between excitation and emission range).

If you swap the filter then it may be best that you test the settings with multipeak (6-8) beads such as our Rainbow Calibration Particles and look at their spread with the original filter and recheck the bead spread with the changed filter.

BioLegend does not test for pathogens in-house aside from the GoInVivo™ product line. However, upon request, this can be tested on a custom basis with an outside, independent laboratory.

Yes

This non specific binding should be kept in mind. You can try testing the antibody first to see how much non specific binding you see in your sample. It is also best to follow routine steps such as Fc blocking and titration of the antibody in order to reduce background while doing flow cytometric staining. You can also consider our True-Stain Monocyte Blocker™ to see if it improves staining (Cat. No. 426101).

Annexin-Phosphatidylserine binding is lost below pH 5.2 and with prolonged incubation over a temperature of 42°C.

If you are not looking for a specific clone check we recommend to use the most popular clone based on Pubmed or Google Scholar.  This will give you an idea on how often a clone is used compared to another.

For detecting human FOXP3, both 259D and 206D clones give comparable staining based on in-house testing. Please see the Treg homepage for the differences between clone 206D or 259D. 206D recognizes human FOXP3 epitope in the region of amino acids 105-235. The 259D antibody recognizes human FOXP3 epitope in the region of amino acids 105-235. For other mammalian species, 150D is recommended. Learn more with our Treg webpage.

Please refer to our Cell Marker webpage . For a more streamlined approach, check out the Essential Markers webpage. It is also advisable to consult the literature to find out further information for a particular cell type.

Diluting the antibodies to perform the staining would also dilute the azide to a concentration likely not to be toxic to the cells. Since the cells are left in contact with the antibody (and the azide) for only 1/2 hour then washed, the there will be little or no residual azide in the sorting buffer, so the chances of residual toxicity are very low.

Intracellular cytokine staining for flow cytometry represents a snapshot in time and ELISA represents cytokine accumulation over time.

Typically, mouse CD206 surface level is relatively low under normal conditions and so intracellular staining protocol is required to get better signal.

We don't test this in house. Typically the molecular weight of an IgG antibody is approximately 150kd.

Most failures of IL-17 intracellular staining are due to stimulation problems. PMA/Ionomycin in liquid form is very unstable.  We recommend storing single use aliquots at -20°C degrees. The aliquots should be thawed at ambient temperature and diluted in PBS to a working concentration immediately before stimulation.

For clone 1412: This clone specifically recognizes cytoplasmic domain of CD20 and thus can only be used for intracellular flow cytometry. In this instance you will need to include the fixation and permeabilization steps. Please follow the intracellular flow cytometry staining protocol.

For clone 2H7: This clone is ok for regular surface staining for CD20 and there is no need for any fixation and permeabilization steps.

Our technical protocols can be found here.

The test size products are pre-titrated for optimal staining of 1 million cells in 100 µl volume.  On the other hand, µg size products are at a defined concentration regardless of the optimal usage.  It is still really important, regardless of format, to use the correct isotype control at the same amount (µg) as their antibody of interest.  If the concentration of the antibody is not on the vial, then call us or email (by clicking on “More Info” below) to obtain the concentration for your lot of product.

All PE, APC and their tandem dyes labeled antibodies have a F/P of 1:1.  Other formats such as FITC or Alexa dyes have various F/P ratios, typically within range of 3-7

It is lot-specific. On average it ranges between 2-4.

It is best to follow protocol as described on the product data sheet. Moreover, RPMI 1640 has a relatively high concentration of phosphate and low calcium ion concentration, which negatively impacts Annexin binding to its target phosphatidylserine (PS). Measurement of cell death by using Annexin V may also be significantly affected by time of incubation on ice, calcium concentration, and type of medium.

It is recommend to perform a titration because sometimes you find you can use less antibody for your own experimental needs, and also to have a feel for how the antibody behaves in your own hands in your particular experiment.

In general our antibodies can be used for cell sorting, but we do not routinely test this in house.  Please note the fluorochrome conjugates are not endotoxin tested and contain 0.09% azide. Typically this concentration is low, so the azide amount will not affect viability of the cells.  As for activation of cells, typically the incubation time with the antibodies for staining is short (~20minutes) and this should not cause activation.  Incubation of antibodies with cells at 4°C or on ice will help prevent activation.

Yes, you can use less cells or less volume per test, as long as the antibody concentration does not change.  For example, if you decrease the staining volume from 100 to 50 µl, you could use half the amount of antibody.  But, if you decrease your cell numbers from 1 million to 100,000 cells, and your staining volume is still 100 µl, then you should still use the data sheet recommended amount of antibody.

Since tandems can exhibit variation between sources and lots, it is best to use the exact same antibody as a compensation control. It may be best to use compensation beads for setting up the compensation.

If the product web sheet for that clone does not reveal this information then it is possible that we have not tested the fixation compatibility of that particular clone. Clone specific literature search is advisable in such situations. Visit our webpage to learn more.

It is not recommended. It is best to use PBMCs for this testing.

The larger holes created by the nuclear permeabilization required for FOXP3 may allow cytokines to leak out of the cell, making it harder to detect lowly-expressed cytokines. You may have to use a control where the cells are only permeabilized through the cell membrane.

It should not be frozen as it will lead to loss of biological activity due to dimerization.

We do not recommend freezing our antibodies as this can denature the antibody or cause fluorochromes to uncouple from the antibody during freeze/thaw. In addition, the antibodies can clump and form aggregates, allowing it to bind nonspecifically to cells. We recommend keeping our flow antibodies at 4°C, protected from light.

Due to continuous recycling of many chemokine receptors, it may be worthwhile to consider staining at room temperature or at 37°C if the staining at lower temperature (which can potentially reduce receptor turnover) is not optimal.

According to a study (Asselin-Labat, ML et al. 2007. Nat. Cell Biol. 9:201) expression levels of CD61 may vary with age as well as pregnancy stages. It may be best to test CD61 on 4-8 week old mice.

Generally, we test  4 to 6 dilutions with the most commonly used target cells (if they are available) for the titration curve, then determine the optimal concentration based on the S/N (Signal/Noise) ratio.

FC is for cell surface staining and ICFC is for intracellular staining.  Yes, the concentrations can be different. The IC antibodies and isotypes have optimized fluorochrome conjugation properties to give optimal performance for IC staining.

Brilliant Violet™ is a family of highly fluorescent polymers, excitable by the 405 nm violet laser, created by Sirigen based on Nobel Prize winning chemistry. There are seven fluorophores available: BV421™, BV510™, BV570™, BV605™, BV650™, BV711™, and BV785™. Each has a unique emission spectra as well as a unique set of advantages over other 405 nm excitable fluorophores on the market. Brilliant Violet™ fluorophores are suitable for surface or intracellular flow cytometry, providing excellent signal with very little background. For excitation/emission and beta testing data, recommended filters, and comparable flourophores, as well as guidance on the strengths of each member in the family and incorporating them into multicolor panels, see our dedicated Brilliant Violet™ page.

We recommend using the standard 450/50 filter to detect Brilliant Violet 421™, which is the same filter used to detect Pacific Blue™.  We recommend using the 510/50 filter to detect Brilliant Violet 510™. For Brilliant Violet 570™ we recommend the 585/42 bandpass filter, commonly used for Pacific Orange™. For BV605™ detection, we recommend the standard Qdot® 605 filter, 610/20 with a 595LP dichroic. For BV650™ detection, the standard Qdot® 650 filter, 660/20 with a 630LP dichroic, is recommended. For BV711™ detection, we suggest the standard Qdot® 700 filter, 710/50 with a 685LP dichroic. For BV785™ detection, we suggest the standard Qdot® 800 filter, 780/60 with a 750LP dichroic. See the Fluorescence Spectra Viewer for complete excitation and emission data.

Brilliant Violet 421™ antibody conjugates give an exceptional signal-to-noise ratio, in some cases greater than 10-fold better than Pacific Blue™.  It gives PE-equivalent brightness or better on the violet laser.  Brilliant Violet 570™ is a non-tandem polymer, and provides improvements over Pacific Orange™, AmCyan, and Horizon™ V500. Brilliant Violet 570™ antibody conjugates also provide much improvement over Pacific Orange™, by as much as 6-fold.  Brilliant Violet 605™ and Brilliant Violet 650™ are significantly brighter than eFluor® 605 and eFluor® 650, respectively, by as much as ten-fold, but are not much brighter than Qdot® 605. Brilliant Violet 711™ is significantly brighter than eFluor® 700 and eFluor® 650, by as much as ten-fold, but is not much brighter than Qdot® 705. Brilliant Violet 785™ is similar in brightness to Qdot® 800. Since BV605™, BV650™, BV711™, and BV785™ are non-nanocrystals, they are likely to perform better for intracellular staining and are not affected by fixation.  Learn more…

Yes, Brilliant Violet 421™ is exceptionally bright and photostable, making it an excellent choice for microscopy. View the data.

Contact our sales team for more detailed information regarding your lab’s discounts.

Yes, our ordering system will be able to process orders using the old catalog number as well as the new catalog numbers. However, the old catalog numbers will be phased out, so you are encouraged to transition to the new catalog numbers, especially where orders are generated automatically from your systems.

No, BioLegend does not test Ultra-LEAF™ antibodies by functional assays unless otherwise indicated. Due to the possible complexities and variations of uses of biofunctional antibodies in different assays and because of the large product portfolio, BioLegend does not currently perform functional assays as a routine QC for the antibodies. However, we do provide references in which the antibodies were used for functional assays and we do perform QC to verify the specificity and quality of the antibody based on our strict specification criteria.

No, BioLegend does not test for pathogens in-house unless otherwise indicated.  However, we can recommend an outside vendor to perform this testing as needed.

BioLegend's Ultra-LEAF™ (Ultra-Low Endotoxin, Azide-Free) purified antibodies are specifically designed for functional analyses, providing the most accurate results with minimal negative effects. Ultra-LEAF™ antibodies have an endotoxin level < 10 EU/mg, and GoInVivo™ antibodies are pathogen tested and have endotoxin levels < 1 EU/mg of antibody.

BioLegend antibodies and reagents that carry the IVD or ASR designation are manufactured in a registered GMP lab, while our other products are manufactured under ISO 13485 certification. Both environments provide high quality products. To learn more about our quality standards visit our Quality Control page at: https://www.biolegend.com/QC.

Your  vial has the antibody concentration on the label, in some cases.  If not, contact tech support or use our lookup tool for the concentration of your lot of antibody.   Match your isotype to the concentration of the primary antibody.

Refer to the for listed isotype and format of each primary antibody and match the isotype control appropriately.  For example, if you have a PE conjugated primary antibody with Rat IgG1, k isotype, use PE Rat IgG1, k isotype control Catalog # 400407 or 400408, as your isotype control. For your conveniece, most products have their isotype control listed under their "related products".

Yes, you can since kappa and lambda represent light chains which don't contribute to the background staining.

We typically use a stabilizer for pre-coated plates. The additional washing step is designed to remove these components before you start the assay. If you do not perform the washing, the effect on assay performance is negligible.

Information regarding the antibody clones used in our kits is proprietary. However, the clonality (polyclonal or monoclonal) and host species of the antibodies may be provided upon request.

This is dependent on what targets you are analyzing and the specifics of your study. Ideally, we advise determining the difference between serum and plasma for the targets of interest and deciding on the sample type to be used for quantification. Depending on the targets, there may be a difference in concentrations of the targets between serum and plasma. The most important factor in preparing plasma or serum samples is consistency in preparation to ensure precise measurements. In general, plasma/serum samples should be free of particulate matters, contain no excess lipids, and have no hemolysis.

These types of contaminants will contribute to background, and adversely affect the precision of the assay. The key is to prepare the sample the same way each time. That is, centrifuging samples at the same speed, for the same time, removing the serum or plasma immediately after centrifugation and aliquoting and freezing the samples at the same time. Avoid repeated freezing and thawing cycles.

For our LEGEND MAX™ and ELISA MAX™ Deluxe formats we recommend following the provided protocol. We cannot guarantee kit performance if there are deviations from the protocol. Below are general suggestions if you are using our ELISA MAX™ Standard format:

• Control background noise by increasing the number of washings and soaking time between washes.
• Control assay precision. For example, be more careful and more consistent in your pipetting, use fresh paper towels for tapping plates to avoid contamination of avidin-HRP and increase washing volumes.
• Increase incubation times. Be aware that this typically also increases background so the assay sensitivity may not necessarily increase.
• Concentrate your sample, if possible.
• Use a five-parameter logistic curve fitting method to more accurately calculate sample concentration at the lower end of the standard curve.

In many cases, the sample may simply contain very low to non-detectable levels of the analyte. No matter how you manipulate the assay, you may still not be able to obtain detectable concentrations.

No, we do not recommend using standalone reagents to replace kit components and cannot guarantee the performance of the kit if you replace or combine reagents from different kits/manufacturers.

This will depend on whether your samples are analyzed in duplicate or triplicate. For example, after adding the standards, you can run 80 samples with no replicates or 40 samples in duplicate and so on.

If coated plates cannot be used immediately, they should be sealed and stored overnight at 4°C.

The wash buffer provided in all our LEGEND MAX™ kits is the same and the part numbers on the wash buffer bottles in these kits should identical. For ELISA MAX™ Deluxe and ELISA MAX™ Standard Sets, we provide a recipe for the wash buffer on each kit’s technical data sheet. This recipe is the same for all ELISA MAX™ sets.

You can order more assay diluent using Cat. No. 421203.

If you are using the same clone for both the detection and capture antibodies, the epitope may already be occupied by one of the antibodies and prevent binding of the other. We recommend choosing different clones for your capture and detection antibodies.

Please consult our ELISA troubleshooting guide.

LEGEND MAX™ Kits are ready-to-go, designed for ease-of-use, and minimize requirements for coating the ELISA plates and preparing buffer dilutions. We recommend LEGEND MAX™ Kits for users who are new to ELISAs.

In general, we recommend using PBS (pH 7.4) or carbonate buffer (pH 9.5). The exact coating buffer you choose may be dependent on the cytokine being detected.

Azide is an irreversible inhibitor of HRP.

Intracellular cytokine staining for flow cytometry represents a snapshot in time and ELISA represents cytokine accumulation over time.

If you are using LEGEND MAX™ or ELISA MAX™ Deluxe format, please consult the individual kit’s manual for more information on sensitivity. We do not provide this information for the ELISA MAX™ Standard Sets but in theory, it should match that of the ELISA MAX™ Deluxe format if you use all of BioLegend’s reagents.

BioLegend's LEGEND MAX™ Kits are guaranteed for 3 months from the date of receipt. ELISA MAX™ Sets are guaranteed for 12 months from the date of receipt. For a lot-specific expiration date, refer to the box label on each product.

TMB Substrate Reagent Set (Cat. No. 421101) contains two components, which need to be mixed immediately prior to use. The TMB High Sensitivity Substrate Solution (Cat. No. 421501) contains everything in one solution with special stabilizers. It is a high kinetic substrate solution which generally gives higher signal. The type of substrate used for an assay can affect the optical density (OD) achievable and the time required to achieve the desired OD.

No, we do not recommend using the recombinant protein standard for any other applications. The protein standards included in the kit are calibrated for use as an ELISA standard and have been not tested for bioactivity. Additionally, ELISA protein standards may contain other carrier proteins and have not undergone the same sterility testing as our bioactive recombinant proteins.

No. Tissue culture plates are designed for cell culture purposes and do not typically have high protein-binding capacity. For ELISAs, we recommend using high protein-binding plates such as Nunc Maxisorp™ plates (Cat. No. 423501).

The antibodies used in our ELISA Kits have been validated for their use in ELISA assays and we cannot guarantee that they will work in other applications, like flow cytometry. Instead, we recommend you use flow cytometry validated antibodies for these experiments.

In general, BioLegend’s ELISA products can be used for tissue or cell extracts/homogenates as long as the samples are prepared in such a way that they are compatible with immunoreactivity. For this, we recommend using the following guidelines:

• Tissues/cells should be lysed or homogenized in a neutral pH buffer that contains no denaturing chemicals (such as urea, thiourea, SDS).
• No or minimal levels of detergent (such as SDS, Triton™ X-100).
• No excessive ionic strength (salt concentration greater than physiological ionic strength).
• The buffers should contain sufficient protease inhibitor cocktails to preserve the target proteins from proteolytic degradation by enzymes released from cells.

We do not recommend reusing samples. For accurate results, samples should be aliquoted and stored for one-time use only. If you decide to reuse a sample, you would need to perform additional validation experiments to ensure you are able to get accurate readings. This would be influenced by a number of factors including sample stability.

If the antibodies were provided as part of a kit, we do not recommend combining reagents from different ELISA Kits. We cannot guarantee the performance of the kit if you combine reagents from multiple kits or manufacturers. If you have purchased individual antibodies and are developing your own ELISA, it may be possible but would need to be tested. Antibodies may not recognize or show poor binding toward the protein standard if the immunogen used to generate those antibodies is different from the protein standard in terms of sequence or structure.

It is best to follow the recommended protocol without any additional delays. However, if you wish to pause the experiment, we would recommend delaying the procedure at the blocking step (after coating with the capture antibody). You can add 200 μL of blocking buffer in the wells and keep the plates overnight at 4°C (minimal or no loss of signal) or frozen at -20°C for few days (it may have some impact on the signal). Delaying the procedure at the first step (coating with capture antibody beyond 16-20 hrs. at 4°C) is not advisable because it may lead to high background.

It may be possible to purchase some kit components individually. Please contact our technical service group and provide lot information of the kit and its components to determine whether this is feasible.

This is not recommended. The ELISA MAX™ reagents are optimized for a particular lot, and they have not been quality tested for applications other than ELISA.

Generally, coating for 16-20 hours at 4°C is recommended. Longer incubation time may increase the amount of capture antibody bound to the plates and this may increase the background noise.

We don’t recommend this practice. Sensitivity of a kit depends on the individual components and their collective validation as a kit and it will not change by adding extra points to the standard curve.

While this may be possible, you may end up with a signal plateau at higher concentrations of the standard. It is generally recommended to use the concentration range recommended in the user manual.

The antibodies in our IL-1β kits were generated against the mature form of the protein. However, because the pro-form of the protein contains the mature form, the antibodies should detect both forms.

No, phenol red in cell culture media will not interfere with the readings from an ELISA assay.

In some cases, dilution with assay buffer is required to minimize the matrix difference between the samples and the standards to achieve better accuracy.

• Increase incubation times (with samples, detection antibodies, or avidin-HRP or TMB substrate). Please note, this may also increase the background in your assay and may not increase sensitivity.
• Shake plates during incubation steps.
• Ensure that the standard is completely reconstituted before use.
• Increased washing and soaking in between washings to further decrease background.
• If possible, read the plate at 450-570 (or closest) nm for background subtraction.
• Improve duplicate CV% by controlling pipetting error or washing with larger volume of washing buffer.
• Use a 5-PL or 4-PL curve-fitting method, rather than a linear curve fitting. This is usually performed with a curve fitting software and provides better calculation at the lower end of the curve.

The primary differences between these kits are the reagents included within the kit or set.

LEGEND MAX™: Fully validated and ready-to-use kits. They contain all required reagents, including a 96-well strip plate pre-coated with capture antibody.

RAPID MAX™: cut assay time down to less than 90 minutes. Each kit is fully validated and contains a 96-well strip plate that has been pre-coated to immobilize the capture antibody.

ELISA MAX™ Deluxe: A more cost-effective option that includes most buffers, pre-titrated antibodies, recombinant standard and substrate. Plates are not included in this kit and must be purchased separately.

ELISA MAX™ Standard: Provides the basic components to complete an ELISA including a recombinant protein standard, capture and detection antibodies, and Avidin-HRP. This set requires some optimization and is perfect for those that want to stretch their budget by providing their own buffers and plates.

No, ELISA MAX™ Deluxe Sets do not come with plates. Coating Buffer and Assay Diluent (for blocking and dilutions) are included in these sets. Plates can be ordered separately (Cat No. 423501), and instructions for coating the plates are in the sets’ product manuals.

Different manufacturers often use different antibody clones in their kits. The specificity of the antibodies partially dictate how much signal is being detected.

Additionally, different kits may use recombinant protein standards that were expressed and purified using different methods. For example, recombinant proteins expressed in E. coli can show different immunoreactivities primarily due to refolding inconsistences. Kit standards are produced and calibrated against different references between manufacturers, making it difficult to compare components from different kits. Each BioLegend ELISA Kit was developed and validated with reagent concentrations and protocols optimized for analytical robustness. Any changes to the reagents (standards, antibodies, matching matrices) and protocols will affect the final assay performance.

It is very difficult to obtain exactly the same sample concentrations at pg/ml levels when comparing different cytokine kits from different vendors, in part, because suppliers may use different reagents (including antibody clones) that have different immunoreactivity toward the target protein. It is not uncommon that the cytokine concentration may vary a few-fold between kits.

In general, particularly for those cytokines in pg/mL ranges, it is more important to have matching biological trends instead of matching absolutely concentrations. This is to say, the same cytokine measured by different kits from different vendors should show the same pattern of biological changes for relevant treatments.

Since every sample is unique, it is difficult to predict as this may depend on the sample preparation and the nature of the analyte. If you are using a LEGEND MAX™ Kit, please refer to the respective manual for more information.

Do NOT invert the plate to dump the liquid. The beads will not adhere to the plate. After centrifugation using a swing bucket rotor with a plate adaptor, you can remove the liquid by using a pipette.

LEGENDplex™ assays can be used on most commonly used flow cytometers that can read APC and PE, such as BD FACSCalibur™, BD FACSCanto™, BD FACSCanto™ II, BD LSR I™, BD LSR II™, BD LSRFortessa™, BD FACSAria™ I, II, III, BD Accuri C6™, BD FACSVerse™, Gallios™, CytoFLEX™, Moflo XDP™, Attune™NxT, NovoCyte™, MACSQuant®, and Guava® easyCyte. Please refer to the “Materials to be provided by end-user” section of your LEGENDplex™ manual for details on the requirements of the machines in terms of laser and channel configurations. For other brands of flow cytometers the end-user needs to make sure that the machine is set up properly before use.

Filter plates or V-bottom plates have been included in some kits for your convenience. Performing the assay with filter plates is generally recommended. A vacuum filtration unit is required to work with the filter plates. If however, you don’t have access to a vacuum manifold, then you can use the V-bottom plates or micro-FACS tubes as described in the manual and follow the recommended assay protocols for the type of plates or tubes you choose. All tubes and plates should be made from low binding polypropylene. Polystyrene ELISA or cell culture plates should not be used.

Yes, all of the above sample types may be used with LEGENDplex™ kits as long as they are prepared in a manner compatible with immunoassays. For tissue homogenate samples, they should be prepared in a neutral pH buffer containing no denaturing chemicals (e.g. SDS, urea, thiourea, cholate, etc.) and no ionic detergents and minimal amounts of non-ionic detergents (e.g. NP-40) may be used in sample preparation. The tissue/cell homogenization buffer should not contain excessive ionic strength above physiological concentrations of salts. If possible, the buffer should also contain protease inhibitors. In addition, the samples should be centrifuged to remove particulates.

If a single laser instrument is used for both reporter (FL2 or PE) and classification (FL3), then compensation is needed to resolve the signal spillover from FL2 to FL3, and vice-versa. Even on dual laser instruments, some compensation may be required. Please check your machine for the need for compensation and when required use the calibration beads provided and follow the Flow Cytometer Setup instructions in the LEGENDplex™ manual. In addition, adjust the PMT voltages such that there is good bead separation, the bead populations are not out of scale, and the PE signal is appropriately amplified to ensure proper signal sensitivity.

LEGENDplex™ and Luminex® are both bead-based multiplex immunoassays that use the basic principles of sandwich immunoassays. Both systems use fluorescence-coded beads to achieve multiplexing. The major difference is in the data acquisition. LEGENDplex™ uses common lab flow cytometers for data acquisition, whereas Luminex®-based assays have dedicated machines for this purpose. Therefore, one of the advantages is that LEGENDplex™ can be run on widely available flow cytometers without needing specialized machines. As such, LEGENDplex™ cannot be run on Luminex® machines.

The LEGENDplex™ data analysis software uses a default fiveparameter logistic curve-fitting algorithm, which determines sample concentrations and calculates minimum and maximum detection concentrations of the standard curve. If the default algorithm does not work for a particular set of data, other curve fitting methods are available in the Options of the software. If sample concentrations fall outside of the maximum detectable range, the sample will have to be diluted and re-analyzed.

The samples can be kept overnight at 4°C while being protected from exposure to light and be read the next day. There may be a decrease in signal, but overall, the assay results should not be affected. Storing the samples for extended periods of time is not recommended, as it could lead to further reductions in signal.

LEGENDplex™ kits are valid for 6 months from the date of receipt. 

Typically flow cytometers generate output files in FCS format (e.g. FCS 2.0, 3.0, or 3.1) and in some cases in list mode file format (LMD). FCS files can be analyzed by different software. Data generated from using LEGENDplex™ kits can be analyzed using the LEGENDplex™ data analysis software freely available on the BioLegend website (biolegend.com/legendplex/software). A free of charge software license dongle is currently provided in the kit. Please check our website for the most updated versions of the software.

Yes. It is possible to use the non-linear part of the standard curve for calculation of results. The LEGENDplex™ data analysis software uses a five-parameter curve-fitting algorithm, which determines the minimum and maximum detection concentrations of each target and reports them. If sample concentrations fall outside of the maximum detectable range, the sample will have to be diluted and reanalyzed.

Yes. We now have software for both Macs and PC. Get more details and download the software at: www.biolegend.com/legendplex/software.

We have found that fixing/sterilizing in samples 1-4% paraformaldehyde in 1X PBS prior to reading the samples out on a flow cytometer is compatible with our LEGENDplex™ reagents. However, incubation time with PFA should not exceed 10-15 minutes. Immediately following the fixation, the PFA should be washed out using the 1X Wash Buffer supplied with the kit. Beads should be resuspended in the 1X Wash Buffer supplied with the kit. Prolonged incubation of beads with 1-4% paraformaldehyde will decrease the signal dramatically.

Zombie dyes have been tested against other leading competitors' fixable viability kits and given comparable results. We also highly recommend that you titrate down the amount of each dye used in order to best match the negative signals of your unstained sample and MFI- (mean fluorescence intensity) stained samples.

It is made in E. coli, covering human aa Met1-Asp320.

These dyes bind in equilibrium with DNA. Therefore, external dye concentration must be maintained during analysis and the dye should not be washed out.

Yes, most fixation reagents are fine to be used with Zombie dyes. However, it should be noted that Zombie dyes can still be sensitive to reactive oxygen species. Light exposure or reagents with hydrogen peroxide can lead to free radical formation, affecting fluorescence.

Zombie dyes that have been tested for microscopy applications in-house will display data on the product webpage. It should be noted that Zombie may not work for dead cell discrimination in every microscopy application, as a complicated point will be to determine the level of Zombie signal that constitutes a dead cell. Another difficulty may be finding the proper plane for microscopy in order to observe the dead cells.

The fixation process can contort and alter the membrane of cells, effectively rendering them as dead. Since the ability of Zombie to stain dead cells is correlated with cell permeability, your results may no longer be a valid representation of dead versus live cells.

Yes, Zombie can be used with Annexin V to discriminate live, apoptotic, and dead cells. Cells double positive for both Zombie and Annexin V are dead, while Zombie-dim/Annexin V-positive cells are apoptotic. Live cells will be Zombie-low and Annexin V-negative. The advantage to Zombie over PI and 7-AAD is that you can now fix and/or permeabilize the cells to stain for cell surface and intracellular antigens.

While we typically do not test Zombie Aqua™ with the UV laser, its excitation peak suggests it is effectively excited at 355 nm. However, we would not recommend using BV510™ off the violet laser and Zombie Aqua™ off the UV laser at the same time. Due to cross-beam excitation of BV510™ by the UV laser and the violet excitation of Zombie Aqua™, this would lead to significantly increased background and excessive compensation requirements.

No, not currently.  We have found that cells are functional without the need to detach the magnetic Nanobeads.

The particles are stored in a neutral pH solution containing BSA and sodium azide.

Hydrophilic polymers.

The average diameter is approximately 130 nm.

1 to 2 years depending on product type.

Yes, they can be sterile filtered as the particles are smaller than 0.22 µm.

Not recommended, however lyophilized particles can be made available as a custom product.

We have not tested this application yet.

There are no restrictions for negatively selected cells, as they are untouched by antibodies. For positive selection, there may be clones that are less efficient than others due to possible epitope competition. Please refer to the Technical Data Sheet or contact the Technical Service Group for advice.

We have tested mouse CD4 positive cells that have been isolated from the spleen and lymph nodes with directly conjugated Nanobeads, as well as CX3CR1 expressing cells from mouse bone marrow. The cells appear unaffected as assessed by migration, differentiation and stimulation assays.

Yes, this is possible, but if you are staining for the same marker as used for sorting, we would recommend using a different clone with non-overlapping epitopes.

We have not determined this yet.

Yes, we do. Please contact our Custom Solutions Team at cst@biolegend.com.

No, not currently.

It is possible that other magnetic separation systems can be used. To learn more about this please contact our Technical Service Group.

The diluted, 1X MojoSort™ buffer contains 1X phosphate buffer saline (PBS) supplemented with 2 mM EDTA and 0.5% BSA. The 5X solution is sterile. To preserve sterility dilute with sterile water under sterile conditions.

The commonly used applications are western blotting, immunoprecipitation and protein purification. These can also be used for immunofluorescence microscopy and flow cytometry.

It is likely the target protein was not tagged or the tag is out of the reading frame or the protein is degraded.

1. the early termination of the translation of epitope-tagged protein;
2. non-specific detection with anti-epitope tag antibody due to no/poor blocking of the immunoblot.
3. partial degradation of the protein
4. the concentration of the primary or the secondary antibody is too high.
5. Washing between the incubations is not efficient

• Tag can be easily and rapidly added to a known gene.
• Multiple tags can be added if required.
• Well-characterized antibodies are available.
• The antibody is specific to the tag, therefore cross-reaction with other proteins is avoided
• Proteins and protein complexes can be purified using standardized practices.
• Tagged proteins can be distinguished from otherwise identical untagged proteins
• Possible to study novel and poorly immunogenic proteins.

A few can be as follows

• A cloned and characterized gene or cDNA must be available
• The epitope tag may interfere with protein structure or function
• Epitope-tagged genes can be expressed at abnormal levels due to the use of heterologous promoters
• The epitope-tagged gene must be introduced into the cell, tissue, or organism of interest.

A lack of signal following western blotting may indicate a few different problems.

1. No or very poor transfer. This can be addressed by quickly checking the membrane with Ponceau-S staining.
2. The expression level of the epitope-tagged protein may be too low. Load more ug quantity of the sample and include a positive control.
3. A very diluted antibody may be the problem, try a few different concentrations of the antibody to probe the western blot.
4. Also, a remote possibility is that the epitope tag is out of frame and is not expressed resulting in a lack of any signal.

Once the products are available on BioLegend's website, contact BioLegend's technical service team for any product questions.

We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!

Most of our carrier-free recombinant proteins are shipped in liquid form, so there is no need for reconstitution. If you need to make dilutions, refer to the formulation on the product datasheet. Stock solutions should be prepared at no less than 10 μg/mL in buffer containing carrier protein such as 1% BSA or HSA or 10% FBS (for chemokines, use either BSA or HSA). For long-term storage, aliquot into polypropylene vials and store in a manual defrost freezer. Avoid repeated freeze/thaw cycles.

For reconstitution of our lyophilized recombinant proteins (ELISA Std, animal-free, and some carrier-free) please refer to the Certificate of Analysis and/or Technical Datasheet included with the product and follow the instructions. 

Our animal-free products are proteins that go through the entire production process without touching any animal containing components. This includes using animal-free media and purification equipment that are animal component-free. Some studies which are particularly sensitive to contamination by mammalian pathogens may require the use of animal-free products. Our carrier-free products do not contain any carrier protein in the solution, as expected, but they are produced using animal-containing components. Both versions are expected to have similar activity and function, though specific activity is lot-dependent.

BSA is used as carrier proteins to improve the stability of the reconstituted protein, and to avoid the product from sticking to the wall of the vial.

The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.

All our carrier-free and animal-free formats of recombinant proteins do not have any additional carrier proteins such as BSA in the formulation. Typically our ELISA standard recombinants have carrier proteins added to the formulation for added stability and to avoid the product from sticking to the wall of the vial. When the presence of carrier is not desirable (e.g., in-vivo applications), carrier-free proteins can be used directly. When carrier proteins do not affect the outcome in a study, the customer can decide what type of carrier protein they would like to use and whether it is necessary to add it to their stock.

Antibodies used are different in different kits. The specificity of the antibodies partially dictate how much signal is being detected.
Recombinant standards used are different.  First of all, different kits may use recombinant proteins expressed and purified using different method. Second, recombinant proteins expressed from E. coli from the same source can show greater than 10 fold difference in term of immunoreactivity from lot to lot, primarily due to refolding inconsistency. Third, different kit standards can be produced and calibrated against different references. So far there is no universally accepted standardization for cytokine immunoreactivities.
Each BioLegend ELISA product was developed and validated with reagent concentrations and protocols optimized for best analytical robustness.  Any changes to the reagents (standards, antibodies, matching matrices) and protocols etc all affect the final assay performance.

Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.

Our carrier-free recombinant proteins are shipped on blue ice.  These products have been validated to maintain activity after shipping using blue ice.

Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.

There is no direct relationship between International Units and the units that are calculated using the inverse of the specific activity because those units are not calculated using the International Standard provided by WHO (National Institute for Biological Standards and control). In those cases, the best way to compare the activity of two sources of recombinants is by doing the bioassay side by side using the same system.  If our protein has been tested using the International Standard, the International Units will be indicated on the technical data sheet for that product.

* Transfer buffers may have become contaminated.
* Post-antibody washes may not have been performed with sufficient time or volume.
* Blocking and incubation agents were not freshly prepared or were too dilute.

*  Transfer efficiency may have been poor. Check protein transfer by staining the gel and/or membrane.
* Incorrect storage of antibodies or ECL western blotting detection reagents.
* Insufficient protein may have been loaded on the gel. Depending on the location of the target protein, membrane or nuclear preparations may be required (instead of whole cell lysates).
* Film exposure time may have been too short.

0.5 μg/ml to 4 μg/ml range can be used for titrations.

We don't test this in house. Typically the molecular weight of an IgG antibody is approximately 150kd.

The size of the target protein can be affected by many factors such as:

• Post-translational modification such as phosphorylation, glycosylation can increase the size of the protein.
• Post-translation cleavage - e.g. some proteins may get cleaved to the active form from pro-protein form via post translational cleavage.
• Different sized proteins produced from the same gene can be produced by alternative splicing (Splice variants).
• Overall charge on the protein based on amino acid composition can also affect the protein migration in a gel.
• Strong protein-protein interactions can potentially lead to Multimers thereby leading to the appearance of higher size bands. This is usually prevented in reducing conditions and boiling of the sample before loading to the gel.

If an antibody is listed as being quality-tested or validated for IHC, we can look up the protocols for you. If this application is reported in literature for IHC, we have not specifically tested the product for this application. It relies on information from publications. Of course, we seek to provide the most updated information on these publications, so we can update our datasheets if the information is incorrect.

Anti-fade should not be added for live cell imaging as it will deprive the cells of oxygen.

No, due to spectral emission overlap you will not be able to distinguish between GFP and AF488/FITC

In most cases, this is true. Antigen retrieval helps both the accessibility of the antibody to the tissue and also counteracts the fixation effects on the recognized epitopes. Check the application references for any additional details for IHC or IF experiments.

Due to their vulnerability to photobleaching, these fluors are not recommended for IF. Dyes such as Alexa Fluor®, DyLight™, and BV421™ are good alternatives for the IF application.

Typical concentrations of monoclonal antibodies for use in IHC are from 5-25 µg/ml. Polyclonal antibodies can be used at a range of 1-10 µg/ml. We will indicate on the datasheet if an antibody has been tested in-house or published for use in this application. In addition, you can do a lit search with the clone name and immunohistochemistry/paraffin/frozen to see what the protocol details are.

There are no special requirements for the peptides. Peptides that naturally bind to MHC molecules will bind to Flex-T™ reagents. For class I molecules, typical length is about 8 – 10 amino acids. Class II molecules accommodate longer peptides, about 14 – 20 amino acids¹.

1) Mohan JF and Unanue ER. Unconventional recognition of peptides by T cells and the implications for autoimmunity. Nat Rev Immunol. 2012 Oct;12(10):721-8.

The T-cell mediated innate immune response is defined by the interaction between antigen presenting cells and T cells, through the Major Histocompatibility Complex (MHC) and the T cell receptor (TCR). MHC molecules present a peptide to antigen-specific T cells that recognize this peptide. Soluble, monomeric MHC molecules bind very weakly to the TCR. However, by making a tetramer through a fluorescently labeled streptavidin conjugate, the complex binds to several TCRs, creating a more stable interaction and making it useful for flow cytometric detection of antigen specific T cells.

There are a number of databases and webpages that can help, these are three of them:

http://www.iedb.org/
http://tools-int-01.liai.org/main/
http://www-bimas.cit.nih.gov/molbio/hla_bind/

Long-wave UV, 366 nm, 8 Watts (We recommend, for example, CAMAG cat# 022.9115, or Ultraviolet Crosslinker CL-1000). The distance from the solution to the light source should be 2 – 5 cm (approximately 0.8 – 3 inches).

Flex-T™ (Flexible-Tetramers) is BioLegend’s brand name for our Soluble MHC product line. It encompasses monomers, the ultraviolet (UV) peptide exchange technology, and all associated products and applications.

The sequence of the UV-labile peptide is not needed to use the reagent. MHC molecules are not stable without a peptide, so these peptides are used just for two purposes: stabilize the MHC molecules and serve as a place holder to be substituted by the peptide of interest.

Not currently.

Yes, please contact our Custom Solution Team with your request at cst@biolegend.com, or contact your local BioLegend representative.

Follow the protocol for HLA class I ELISA. An assay positive control is provided (Cat#280301) that can be diluted to a high, medium, and low concentration. Signal intensity can be correlated to affinity of the peptide.

Flex-T™ MHC monomers are loaded with a peptide that can be degraded by the use of a UV light source. This allows for a peptide exchange when the UV irradiation is done in the presence of the peptide of interest (which is not UV-labile). This flexibility permits the screening of virtually any peptide of interest with enough affinity for the MHC allele that it is loaded onto.

Please see the linked document titled “Peptide Sequences”. The document contains a table with commonly used peptides by allele to study antigen specific T cells.

With the traditional pre-assembled Tetramer approach, this is difficult to do, and not cost-effective. With Flex-T™ technology, as there is more flexibility to assemble the tetramers, it is easy and affordable to screen a sample for several specificities². To facilitate this approach, a combinatorial color coding system has been developed. Please visit the Flex-T™ webpage for a detailed description.

2) Hadrup SR et al. Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers. Nat Methods. 2009 Jul;6(7):520-6.

Yes, the same monomer can be assembled with different Streptavidin conjugates, providing great flexibility for color choices. Additionally, it can be stored frozen and the tetramer assembled shortly before the experiment. This increases the storage time of the reagent.

Depending on application pursued it may require diluting the antibodies prior to use.

They are treated with our proprietary mixture prior to lyophilization.

Yes, it should be possible, although this has not been verified by BioLegend.

For this, use Veri-Cells™ Activated (Cytokine) PBMC which have been activated with Cell Activation Cocktail (with Brefeldin A) containing PMA and Ionomycin. These have been validated to stain positive for activation-induced cytokines including TNF-α and IFN-γ. Veri-Cells™ PBMC and Veri-Cells™ CD4-Low PBMC will not have detectable TNF-α or IFN-γ.

Each Veri-Cells™ product is tested with a panel of antibodies to ensure expression of these markers falls within the expected range.  For more information regarding the exact markers used for each product, please contact technical service.

You can dialyze or desalt the enzyme into a phosphate-free buffer.

Yes, the reagent is designed for drug screening work and other situations that require DMSO.

This is almost certainly caused by inadequate mixing of the Stabilizer. This results in a high background signal because of non-enzymatic decay of ATP substrate. The Stabilizer is added in a relatively small volume (20μL), and the operation of pipetting up and down with a pipette set to 20μL volume may not result in sufficient mixing when the total volume is 270μL. Try pipetting up and down while stirring at the same time. Alternatively, add the Stabilizer with one pipette set at 20μL volume and mix using a larger pipette set to ~150μL volume. This ensures thorough mixing of the Stabilizer solution with minimal effort.

Purify the DNA from your sheared chromatin samples and run the DNA on a 1-2% agarose gel. DNA should be sheared to a range of 100-900 bp. Below is an example of chromatin from a number of different cells lines that has been sheared optimally using enzymatic digestion compared to chromatin which was over-digested.

Lane 1: Ladder, Lane 2: HeLa, Lane 3: Jurkat, Lane 4: NCCIT, Lane 5: HeLa IL-6	Lane 1: HT1080+ IFNγ, Lane 2: NIH353, Lane 3: HT29, Lane 4: HCT116, Lane 5: Ladder

Sheared chromatin may be saved at -80°C prior to performing the immunoprecipitation. If you are saving the chromatin, we would recommend aliquoting the chromatin as it can be sensitive to freeze thaw cycles. Alternatively, purified DNA can be saved prior to downstream analysis.

Sonication may be ideal for difficult to lyse cell types and provides random fragmentation but may damage epitopes. It is not suitable for Native ChIP experiments (in which the chromatin has not been cross-linked). Enzymatic digestion is milder and can be used in Native ChIP experiments but will exhibit sequence bias and may not be suitable for hard to lyse cells.

Human and mouse cell lines such as HeLa, Jurkat, Daudi, THP-1, NIH3T3, and 293T have been validated using this protocol. Other cell types (plant, yeast, tissue, FFPE samples, etc.) may require optimization.

An important consideration when performing ChIP is the amount of chromatin that will need to be loaded to the column in order to elute sufficient IP’ed DNA for downstream analysis. Sufficient cell numbers should be used so that at least 3µg of chromatin can be used per IP. Start with 1-15 million cells. However, the cell number can be scaled up and the reagents need to be adjusted accordingly.

This should be determined empirically by the end-user. Refer to the datasheet for our Go-ChIP-Grade™ Purified Antibodies, or other ChIP-validated antibodies. The suggested dilution of the Go-ChIP-Grade™ anti-RNA Polymerase II Antibody; Clone 8WG16 (positive control) is 1:300 – 1:500 by volume.

The quality of the ChIP antibody has a major impact on the success of the assay. Use only ChIP-validated antibodies. Make sure to have good quality chromatin samples with fragments between 150-900bp. Insufficient DNA shearing leads to longer DNA fragment length that can cause high background in downstream experiments. Insufficient wash steps can also leave traces of non-specific chromatin alongside enriched DNA. If background remains high, include an additional wash step during the IP protocol.

Make sure to only use ChIP-validated antibodies and follow the instructions for the amount of antibody to be used or titrate the antibody for your experiment. It is also recommended to include ChIP validated positive and negative controls (included in the kit) to validate the efficiency of your ChIP assay. Good quality chromatin samples and chromatin fragment sizes between 150 – 900 bp are also critical. Other factors can include whether cells were effectively lysed, over-fixation and reduced antibody binding due to decreased epitope availability, and insufficient starting material (chromatin sample per IP).

TotalSeq™ is BioLegend’s brand of antibody-oligonucleotide conjugates, to enable simultaneous analysis of proteins and mRNA in single cells. CITE-seq and REAP-seq are two similar workflows for simultaneous protein and mRNA analysis, and the TotalSeq™ conjugates integrate seamlessly into these established protocols.

The main difference between the 3 formats relates to the sequences of the oligo tags and their downstream compatibility with single-cell sequencing platforms.

• TotalSeq™-A reagents use a poly-A capture method that is instrument agnostic which includes the 10x Genomics 3’ Single Cell Gene expression kit.
• TotalSeq™-B utilizes the feature barcode technology for 10x Genomics 3’ Single Cell Gene expression kit.
• TotalSeq™-C also uses the feature barcode technology but for 10x Genomics 5’ V(D)J expression kit.

They each use different primers for library amplification. You can read a little more about the differences on our TotalSeq™ webpage.

We are not currently able to support the mixing of TotalSeq™-A and TotalSeq™-B reagents in a single experiment. These two product lines require different protocols for library prep, and the different capturing methods may cause discrepancies in capture efficiency. In addition, it can often cause issues downstream in the bioinformatics portion when analyzing your data due to the variations in index sequences and lengths. In theory, they should work together, but in practice it can be quite difficult and we advise against doing so whenever possible.

Either will work fine.

When discussing single-cell data, features are any aspect of the cell that is being measured. Common features are mRNA, protein, sgRNA, etc.

TotalSeq™-A/B/C products are fully supported by BioLegend and have been internally tested by BioLegend on the 10x Genomics platform. TotalSeq™-B and C are fully supported by 10x Genomics. 10x Genomics provides limited support for TotalSeq™-A.

If you are unsure about which technical service team to contact if you have questions or issues, feel free to reach out to BioLegend and we will work with our partners to resolve your problem or answer your technical questions.

Hashtag reagents are intended to be used for sample barcoding, which allows users to combine multiple samples into a single lane, then de-multiplex during analysis. Instead of select antigen-specific antibodies, the hashtags are designed so that they are specific for human or mouse cells, and to cover as many cells as possible. For human samples, the hashtags are made of two antibodies that recognize ubiquitous surface markers, CD298 and β2 microglobulin, each conjugated to the same oligonucleotide containing the barcode sequence. For mouse samples, the surface markers are CD45 and H-2 MHC class I. The conjugates are already pre-mixed and ready to use.

Hashing allows customers to run multiple samples in a single lane of a 10x Genomics Chromium instrument, or equivalent, which optimizes the number of cells or samples that can be analyzed simultaneously. This can reduce variability due to batch effects or sample handling. It can also help to identify doublets more efficiently. Furthermore, it can also improve yield and help with cell input when cell numbers are low, therefore optimizing the use of the platform and your workflow or protocol.

Typically, when using a single-cell platform, as you increase the amount of input cells, the rate of doublets (two cells captured as one data point) increases. This leads to an unacceptable percentage of datapoints that contain more than one cell. Hashtags allows you to “super load” the number of cells. To this end, multiple aliquots of the same sample could be stained with as many hashtags as aliquots you’d wish to mix. The number of aliquots and number of cells per aliquot may vary, but after washing and mixing the cells, following the staining and analysis protocol, if more than one hashtag is detected in a single-cell “event”, that event can be safely discarded as a doublet. This does not eliminate doublets that contain the same hashtag but the rate of doublet detection is greatly increased. However, “super loading” should be carefully controlled as the more cells you load the more doublets occur, causing diminishing returns overall. There should be a balance between multi-sample optimization, single-cell data yield, and optimal instrument/platform performance.

Nuclear hashtags are used for single-nucleus RNA-seq (snRNA-seq) samples. snRNA-seq captures RNAs that are isolated with the nucleus. This is done because whole, intact cells may be difficult to isolate due to specific experimental or sample conditions, or to answer a specific scientific question. See an example of nuclear hashtag application in this paper: https://www.nature.com/articles/s41467-019-10756-2

Unfortunately, our TotalSeq™ reagents are not directly compatible with single ATAC-seq kits at this time. However, the NYGC has pioneered a bridging method ATAC with Select Antigen Profiling by sequencing (ASAP-seq) which is able to bridge TotalSeq™ antibodies with other capture platforms such as scATAC-seq. https://www.biorxiv.org/content/10.1101/2020.09.08.286914v1

Our nuclear hashtag products available off the shelf, but we do not currently have a recommended protocol for this application at this time.
The only recommendations we can provide are literature references such as this one:
https://www.nature.com/articles/s41467-019-10756-2

Hashtags can absolutely be used in any single cell platform including the 10x Genomics platform. BioLegend fully supports the usage of hashtags. Please contact our technical service group for more information.

We have not tested this, but there is no reason to believe that non-competing clones for the same target would be problematic. Similarly, a sub-saturating concentration of both reagents against the same target should be tolerated by the cell/technique. The original CITE-seq paper by Stoeckius et al.(Fig. 2) demonstrated that cells can be co-stained for downstream analyses. Although the authors did not use current TotalSeq™ reagents, the technology is the same. In addition, other CITE-seq users have also demonstrated this approach. Please contact our technical service group for more information.

We recommend >95% viability before beginning you CITE-seq experiment. Running dead cells during CITE-seq essentially leads to “wasting” single cell runs on dead cells, which may ultimately lead to sub-optimal data, or not processing enough cells to pick up the positive events, especially if the frequency of your target cell is very low. Dead cells can also be removed using magnetic beads. If performing CITE-seq before eliminating dead cells is required, it is possible to use bioinformatics methods to try to clean up low quality events (which may include dead cells). This could be a more complex approach, but in such cases, we recommend the following reading:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758103/
https://www.ncbi.nlm.nih.gov/pubmed/28045081

If the populations of interest can be clustered/identified without sorting, the likelihood that you need to pre-sort is minimal. However, if the cell number in the cluster is very small, it may not be sufficient to draw conclusions when compared to control, or other cell populations. In which case, enrichment of this population prior to CITE-seq analysis may help with obtaining more meaningful sequencing data that is selective for your rare cell population(s) of interest. We also recommend to sort out dead cells if the viability in a sample is lower than 95%.

We have developed pre-titrated, lyophilized TotalSeq™ panels to facilitate the use of combined antibodies. We have panels in all three formats (TotalSeq™-A, B, and C) designed to identify the main immune cells, as well as a universal panel that contains antibodies against 130 protein targets, and 7 isotype controls. We will keep on releasing these pre-optimized panels as we understand that titrating dozens of antibodies for this application is not an easy task. We may not be able to provide specific titration values for individual antibodies as that may depend on multiple factors. However, here are some recommendations in case you may not be able to use our panels, and need to perform your own titrations:

1. Performing titrations using the actual CITE-seq method is costly, but using hashtags can make the process more affordable. See Fig. 3 and associated methods of this publication for more information.
2. Perform flow cytometry experiments to find the optimal antibody concentration, using the same clone conjugated to PE. The flow cytometry antibody amount should translate well to CITE-seq.
3. Label the cells with different concentrations of a TotalSeq™ antibody, and then use a poly(dT) oligo conjugated to a fluorophore, such as Alexa Fluor® 647, as a secondary reagent to detect the TotalSeq™ antibody. This is a more direct method, but it requires the use of the actual TotalSeq™ antibody.

Some available resources include CITE-Seq-Count, and the tools developed by the Rahul Satija laboratory. Please contact our technical service group for further information.

There are several companies offering primer synthesis. We can recommend IDT technologies. For additive primers, desalt purification is sufficient. For index primers, either HPLC or PAGE is needed.

Separate ADT/HTO and mRNA libraries provide flexibility when sequencing. Libraries can be mixed at different ratios before sequencing depending on the number of reads needed. Typically, ADT/HTO libraries require less sequencing depth compared to their mRNA counterparts.

The oligo is attached to the antibody in the same method as some of our fluorophore-conjugated antibodies that are quality tested by flow cytometry. Once the antibodies have been conjugated to the oligonucleotide, we verify by flow cytometry that the antibody can still bind to its target. This is part of our quality control process for TotalSeq™ conjugates.

We recommend to thaw in warm water after removing from the fridge.  The Brefeldin A is dissolved in DMSO, which freezes at 18°C, so this is normal.  No one has reported any problems with the freeze/thaw cycles and this is how we recommend it is stored.   If customers are concerned, they can aliquot the Brefeldin A to avoid the freeze thaw.

We would not recommend extended storage of the product after dilution.  It is best to prepare the dilution as needed on the day of use.  Because there are no preservatives in these buffers and they could be susceptible to changes in pH, long term storage greater than a day or two is not recommended.

FluoroFix™ (cat# 422101) is designed for fixation of cells stained with fluorescent labeled antibodies, including tandem dyes.

Annexin V binding requires the presence of calcium in the solution.  So, we provide Annexin V Binding Buffer (cat # 422201), which is optimized for the best performance of Annexin V staining.

Brefeldin A is provided at a 5mg/ml concentration.

Monensin is provided at a 2mM solution.

Keeping stained cells in any fixative for longer periods of time is not recommended. You can use Fluorofix™ buffer as recommended on the data sheet, followed by washing with cell staining buffer and then at the last step, resuspend your fixed cells in cell staining buffer till you acquire your samples in a day or two.

It is not recommended. It is best to use PBMCs for this testing.

Generally, Brefeldin A is more toxic for longer term incubations, so for shorter stimulations (6 hrs or less) use Brefeldin A.  For longer stimulations use monensin.  We recommend optimization for each cell type and protocol.

This may work with lower numbers of total cells, but we recommend trying to keep higher concentrations of cells for faster analysis. Of course, how many cells are needed depends on the specific application. Successful staining has been done with 1 x 10⁵ cells/well.

Lyophilized product has a guaranteed shelf life of 6 months unopened. Reconstituted plates can be used or stored at 2 - 8°C sealed in the dark and used within a month. Be sure to properly seal the plates to prevent evaporation and shield the antibodies from light.

The kit should be stored at 2 - 8°C upon receipt. Once opened, the plates must be reconstituted immediately. Reconstituted plates can be used or stored at 2 - 8°C sealed in the dark and used within a month.

Yes, as long as the fluorophores on these antibodies are compatible and proper compensation has been applied during acquisition and analysis.

The antibody is in a one test per well format. There will not be any antibody left if the full test is used. Customers may decide to use less than the recommended volume per test, but this is not recommended and the performance is not guaranteed. Customers may also selectively transfer certain antibodies from the original plate to a new plate and use after reconstitution. If any antibody is not used after reconstitution, the plate can be sealed and store at 2 - 8°C for a month in the dark. Once reconstituted, re-drying is not recommended as this may result in a loss of signal.

Yes.

Yes. Customers can use half of the plate or whatever specificities they are interested in. However, the whole plate should be reconstituted. The half plate of antibodies must be transferred to another empty plate for the staining. The remaining half must be sealed and stored at 2 - 8°C in the dark and used within a month.

The plates are not sterile. You would handle them as you would handle most typical flow cytometry staining protocols.

For more info, visit: biolegend.com/custom_solutions.

If the protocol is followed closely, the variability should be minimal. The variability should be similar to single vial antibody staining.

Currently, we do not offer any automated analysis solutions for LEGENDScreen™ kits. However, a commercial solution is available from Astrolabe Diagnostics. The Antibody Staining Data Set is an example application of the LEGENDScreen™ Human PE Kit (see Amir et al.).