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.

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: techserv@biolegend.com

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.

For U.S. orders, if products are in-stock, they are shipped the same day, via Federal Express overnight, so you can expect to receive the products the next day. International direct orders are shipped on Mondays and Fridays, and typically arrive within 2 - 10 days, depending on how long they are in customs.

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

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.

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.

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.

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.

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.

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).

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.

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.

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.

For detecting human foxp3, based on our internal testing data, both 259D and 206D clones give good staining. 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. Click Here

In the following publication the authors state 206D clone is the best, compared to other foxp3 antibodies (236A/E7, PCH101). However, they did not compare clone 259D. Click Here

 Both of the antibodies perform well in our hands and we do not have a particular preference. However, in the current literature, scientists seem to prefer 206D. For other mammalian species, 150D is recommended.

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.(per the Purdue Cytometry website)

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.

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.

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

We have tested IFN-γ with FOXP3 staining and it does work.  It is recommended to use BioLegend FOXP3 Fix/Perm Buffer set and use the FOXP3 staining protocol.  The only addition is to add the cytokine antibody at the same time as the FOXP3 antibody.  In order to capture cytokines intracellularly, cells should be stimulated in the presence of monensin or brefeldin A. The stimulation protocol available on our website is a useful reference for generating various cytokine producing target cells.

BioLegend antibodies are offered in the following formats/ applications:

Format   Application
Purified - FC, ELISA Cap., WB, IP, IHC, IF
LEAF™ - Purified Bioassays, ELISPOT Cap., Blocking/Neutralization, Activation, Costimulation
Biotin -  FC, ICFC, ELISA Det., ELISPOT Det., IHC, IF
Brilliant Violet 421™ - FC, ICFC, IF
FITC -  FC, ICFC, IF
PE -  FC, ICFC
APC -  FC, ICFC
PE/Cy5 -  FC
PE/Cy5.5 - FC
PE/Cy7 -  FC
APC/Cy5.5 - FC
APC/Cy7 - FC
Alexa Fluor ® 488 - FC, IF
Alexa Fluor ® 647 - FC, IF
Alexa Fluor ® 700 - FC
Pacific BlueTM - FC, IF
PerCP -  FC
PerCP-Cy5.5 - FC
Cy3  - IF

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.

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.

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.

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

I would always recommend performing 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.  Most of our products can be used less than what we recommend if the customer does not care too much about the antibody saturation in their staining. the % positive staining should be maintained with concentration selected – ie if while they are titrating they find the population % drops then they are not staining all the cells and should use a higher concentration.

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. It should be the one before the S/N decreases meaning we always release the optimal concentration at its saturation (if it is possible). Most of our products can be used less than what we recommend if the customer does not care too much about the antibody saturation in their staining. the % positive staining should be maintained with concentration selected – ie if while they are titrating they find the population % drops then they are not staining all the cells and should use a higher concentration.

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.

Be sure that the isotype control matches the same concentration/amount being used for the primary antibody. Concentrations between isotype and primary antibodies are not always identical. Therefore, using the same volume may not produce equal amounts of antibody.

Please see our stimulation guide: http://www.biolegend.com/media_assets/support_protocol/BioLegend_StimulationGuide_101711.pdf

Brilliant Violet™ is family of fluorescent polymers, excitable by the 405 nm violet laser, created by Sirigen based on Nobel Prize winning chemistry. The first in the series is Brilliant Violet 421™, emitting optimally at 421 nm, which can be used in place of Pacific Blue™, Horizon™ V450, eFluor® 450, and Alexa Fluor® 405.  Brilliant Violet 570™ emits optimally at 570 nm, and can be used in place of Pacific Orange™. BV605™ emits optimally at 603 nm and can be used in place of Qdot® 605 and eFluor® 605NC. BV650™ emits optimally at 645 nm, and can be used in place of Qdot® 650 and eFluor® 650NC. Learn more...

Most Pacific Blue™ antibodies will not be discontinued, and we continue to make them available to customers.  As more customers transition to Brilliant Violet™, discontinuation will be based upon customer demand for products.

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™ 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. Since BV605™ and BV650™ 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 beta-testing data…

Select Brilliant Violet™ antibodies have been beta-tested and verified by over 20 premiere immunology research labs world-wide, in a variety of applications including multi-color flow cytometry, microscopy, tetramer staining, and cell sorting, as well as a variety of instruments, including common flow cytometers and the Amnis ImageStream.  BioLegend QC tests every lot of product pre- and post-bottling.  As with all products, we back every Brilliant Violet™ product with our 100% guarantee.

We recommend using the standard 450/50 filter to detect Brilliant Violet 421™, which is the same filter used to detect Pacific Blue™.  For Brilliant Violet 570™ we recommend the 585/42 bandpass filter, commonly used for Pacific Orange™. For BV605™ detection, the standard Qdot® 605 filter, 610/20 with a 595LP dichroic, works well. For BV50™ detection, the standard Qdot® 650 filter, 660/20 with a 630LP dichroic, works well. See the Fluorescence Spectra Viewer for complete excitation and emission data.

BioLegend's LEAF™ (Low Endotoxin, Azide-Free) purified antibodies are specifically designed for functional analyses, providing the most accurate results with minimal negative effects. LEAF™ purified antibodies are tested for sterility, and endotoxin is <0.01 ng/µg of antibody.

Yes, we do provide discounting based on quantity of antibody requested.  Please contact us to get a quote.

No, BioLegend does not test LEAF™ antibodies by functional assays. 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.  However, we can recommend an outside vendor to perform this testing as needed.

Although BioLegend does not manufacture antibodies in a certified GMP facility, our products are manufactured according to GMP guidelines. For more information, visit our Quality Control page at http://www.biolegend.com/QC.

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".

Your  vial has the antibody concentration on the label, in some cases.  If not, call or email tech support to (click "More Info" below) and ask for the concentration of your lot of antibody.   Match your isotype to the concentration of the primary antibody.

The ELISA MAX™ Standard Sets contain pre-titrated Capture Antibody, Detection Antibody, Avidin-HRP Reagent, and Recombinant Standard.
The ELISA MAX™ Deluxe Sets contain all of the above, PLUS uncoated Plates, Coating Buffer, Assay Diluent, and TMB Substrate Reagent.

LEGEND MAX™ kits with Precoated plates : Fully validated KIT
 Most convenient, analytically and biologically validation provided, shortest protocol
 Ready to use reagents, including Pre-coated plates
ELISA MAX™ Deluxe: 
 cost effective, includes plates, some buffers
 Pre-titrated antibodies, AV-HRP, Recombinant Standard,  PLUS uncoated Plates, Coating Buffer, Assay Diluent, and TMB Substrate Reagent.
ELISA MAX™ Standard: Cost effective development SET
 most cost effective, plates not included
 pre-titrated antibodies, AV-HRP, and Recombinant Standard.
 Optimization required

No, the plates are uncoated, but Coating Buffer and Assay Diluent (for blocking and dilutions) are included in the ELISA MAX™ Deluxe Sets. Instructions on coating the plates are in the Sets' product manuals and website under "Support".

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

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 an official expiration date of each lot of product, refer to the CoA included in each Kit or Set.  

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

This may be dependent on the cytokine being detected, but in general scientists use PBS, pH7.4 or carbonate buffer at pH 9.5.

Azide is an irreversible inhibitor of HRP.

The plates can be coated with the capture antibody overnight at 4°C and stored at 4°C for a couple days.   As for the blocking step, keep the plate in the blocking buffer for overnight or 1-2 days at 4°C (sealed) and continue with rest of the protocol. The plates should not be allowed to dry.

http://www.biolegend.com/media_assets/support_resource/elisa_troubleshooting.pdf

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

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 BioLgend 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.

• Increase incubation times (1st incubation, detection, avidin-HRP or TMB substrate)
• Shake plates during incubation steps
• Make sure 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 570-590 nm for background subtraction.
• Improve duplicate CV% by controlling pipetting error, washing with bigger volume of washing buffer, etc
• Use a 5-PL or 4-PL curve-fitting method for better calculation at the lower end of the curve. This is usually done with a better curve-fitting software, rather than the linear curve fitting.

This is dependent on the targets being detected and the biological processes. Customers are advised to study the difference between serum and plasma for the targets of interest and decide on the sample type to be used for quantification. Depending on 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 in the same time. Avoid repeated freezing and thawing cycles.

Currently, it is very difficult to  obtain exactly the same  sample concentrations at pg/ml levels when comparing  different cytokine kits from different vendors, partly because of following reasons:
• As mentioned in the question above, the immunoreactivity for the reagents vary from different suppliers.
• In the area of cytokine quantification particularly for those cytokines at pg/mL ranges, the general consensus is that it is more important to have matching biological trend instead of matching absolute concentrations.  This is to say that the same cytokine measured by different kits from different vendors should show the same pattern of biological changes predicted for the relevant biological treatments.
•  For some cytokines, it is not uncommon  that  the cytokine concentrations vary a few-fold between kits for the reasons mentioned above.
• It is critical that kits produced by the same vendor  to keep its assay products consistent from lot-to- lot over time.
We tested  ELISA standards from competitor's kits. Results showed clear discrepancies in the relative potency of the standards in term their immunoreactivities.

Please contact services@biolegend.com

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

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.

Carrier-free recombinant proteins do not have any protein in addition to the cytokine, and non carrier-free have a carrier protein. The difference is the recommended application. We recommend using the carrier free format for bioassay as this is how it is tested.  While the “non carrier free” is biologically active, it is tested by ELISA, and the carrier protein included is BSA. It is useful for other non live cell techniques eg ligand blocking for IHC and IF. 

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 BioLgend 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.

* 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.

Typical concentrations of monoclonal antibodies for use in IHC are from 1-10µg/ml.  While we do not test for IHC application in house, if an antibody has been published for use in this application, we indicate this on the datasheet. In addition, you can do a lit search with the clone name and immunohistochemistry/paraffin/frozen to see what the protocol details are.

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.  BioLegend does not test antibodies for IHC, so check the application references for use of the antibody in paraffin-embedded samples.

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

Monensin is provided at a 2mM solution.

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

BioLegend’s FOXP3 antibodies are optimized for use with our FOXP3 Fix/Perm Buffer Set (cat # 421403).  Buffer sets from other vendors may not work adequately with our antibodies.  For this reason, we highly recommend using our FOXP3 Fix/Perm Buffer Set.

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.

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.

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.

Ask us

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.

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