Western blotting is a key tool in life science research, used to separate and identify specific proteins from complex mixtures. BioLegend offers a wide selection of antibodies validated and quality control tested for Western Blot applications. This includes primary and secondary antibodies. Discover our Direct-Blot™ primary antibodies directly conjugated to horseradish peroxidase to streamline your western blot experiments.

Western Blotting Introduction

Western blotting refers to a routine technique used to separate and identify proteins from complex mixtures. Proteins are separated (potentially under denaturing conditions) in a gel by size before being transferred to a membrane. The protein of interest is probed with specific antibodies and detected through a number of means, commonly chemiluminescence.

 

Figure 1. Overview of the western blotting procedure.

 


Sample Preparation Guide

For western blotting, sample preparation is a key step in the western blotting process. Proper sample treatment ensures you are able to detect proteins of interest. Below, you will find the general methodology used at BioLegend. You may wish to consult literature for more specific protocols relating to your samples of interest.

 

For cell culture samples

I. Isolation of cells: Isolate and wash the cells using ice-cold or pre-chilled PBS. For adherent cells, scrape the cells using a cell lifter. For suspension cells, centrifuge suspension to separate the cell pellet.
II. Cell lysis: Lyse the cells using a lysis buffer with added protease inhibitor.
III. Sonication: Sonicate the crude cell lysate to disrupt the highly viscous cellular DNA.
IV. Centrifugation: Centrifuge the sonicated lysate and collect the supernatant.
V. Protein estimation: Protein concentration is estimated using BCA or Bradford assay.
VI. Sample dilution and heat treatment: After protein estimation, dilute the samples with gel loading buffer/sample buffer and heat for 5-10 mins at 95-100°C.

For tissue samples

I. Homogenization: Smaller solid tissues are soaked in ice-cold lysis buffer and homogenized by using an electric homogenizer. For larger tissue samples, a blender is used to homogenize the tissue in PBS followed by treatment with lysis buffer to disrupt the cell membrane.
II. Centrifugation: Centrifuge the lysate and collect the supernatant.
III. Protein estimation: Protein concentration is estimated using BCA or Bradford assay.
IV. Sample dilution and heat treatment: After protein estimation, dilute the samples with gel loading buffer/sample buffer and heat for 5-10 mins at 95-100°C.

 


Electrophoresis

To start a western blotting procedure, gel electrophoresis is used to separate macromolecules in a sample. The most common type of electrophoresis used is called SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis). SDS is a type of detergent that adds a negative charge to amino acids in a protein, this along with heat applied during sample prep disrupts the tertiary and secondary structure of the protein. Because of the SDS, all proteins will have the same negative charge, resulting in separation being based on size rather than charge.

 

Once an electrical field is applied to the gel, small protein molecules move quickly the through the gel matrix toward the positive electrode, while larger proteins move through more slowly, resulting in a series of bands containing proteins of a particular size (Figure 2).

 

Protein samples are run along side a protein ladder containing several standards of known molecular weights. By using a ladder, the size of proteins in the sample lanes can easily be determined. BioLegend offers the Prime-Step™ Prestained Broad Range Protein Ladder, a three-color protein standard with 10 pre-stained chromophore-conjugated recombinant proteins covering a wide range of molecular weights, from 6.5 to 270 kDa.

Figure 2. Negatively charged small protein molecules move  through the gel matrix, toward the positive electrode, more quickly than larger negatively charged protein molecules.


Transfer

After gel electrophoresis separated proteins on the gel are transferred onto a nitrocellulose or polyvinylidene difluoride (PVDF) membrane, again utilizing electrophoresis (Figure 3). The membrane is then blocked with neutral proteins, such as BSA or milk, to prevent non-specific binding of antibodies to the surface of the membrane.

Figure 3. Transfer of the protein bands resulting from SDS-PAGE on to a membrane by electrophoresis


Antibody Binding/Target Detection

It is important to recall that SDS treatment of samples denatures proteins, causing them to lose their native conformation. This is why conformation epitope-specific antibodies and even flow cytometry antibodies may not always work in a western blotting assay. After transfer, the membrane is incubated with primary antibodies that bind specifically to the target protein, the primary antibody is not typically directly detectable. In colorimetric and chemiluminescent detection methods, the membrane is subsequently incubated with a detectable tagged secondary antibody specific to the host species of the primary antibody. Enzyme reporters like alkaline phosphatase (ALP) and horseradish peroxidase (HRP) are then used to generate signal in combination with a substrate like our Western-Ready™ ECL Substrate Kit. Direct-Blot™ products allow users to utilize primary antibodies directly conjugated to HRP, avoiding the need for a secondary reagent and streamlining the western blot process. For fluorescence-based detection methods, fluorophore-conjugated primary antibodies are used. These fluors typically emit in the near infrared range for detection.

 

 

Figure 4. There are multiple ways to detect proteins of interest in a western blot. The colorimetric method detects signal via colored precipitate. In a chemluminescence-based method, light is emitted by the reaction shown. And finally, in fluorescence, a fluorophore-labeled antibody emits the signal.

Although, western blotting is a well-documented immunoassay, perfecting and optimizing your protocol can be tough. There are a number of common issues that may arise when developing new protocols for various reagents. Below you will find a comprehensive guide to help you troubleshoot common issues reported when western blotting.

 

You can also take a look at our recommended western blotting protocol for additional help. Or, contact tech@biolegend.com for more troubleshooting information.

 

Sections:

No or Low Signal

High Background

Multiple or Non-Specific Bands

Diffuse Bands

Smile Effect on Bands

Uneven Staining of Gel

Target Band is Extremely High/Low on Blot

Lane with Protein Ladder is Black

Black Dots on the Blot

BioLegend Western Blot Troubleshooting Guide
What's the issue? Possible Causes  Solution
No or low Signal The primary and secondary antibodies are not compatible. Ensure you are using secondary antibody that binds to your primary antibody (i.e. if your primary is rat, be sure you are using an anti rat secondary).
Insufficient primary or secondary antibody has bound to the protein of interest.  Utilize a higher concentration of antibody and or incubate for a longer period (i.e. overnight at 4°C).
There is not enough antigen.  Load a larger amount of protein onto the gel. Use protease inhibitors and run the recommended positive control.
Overuse of primary antibody. Use fresh antibody (the effective concentration is lowered after each use).
Incubation with detection reagent not sufficient.  Increase the blots incubation time with detection reagent.
Detection reagents are not working. Make sure detection reagents are functional by testing with a different primary antibody.
Poor transfer during blotting. Make sure the transfer apparatus is set up correctly. Ensure you are using the correct transfer times. 
Secondary antibody is inhibited by sodium azide. Do not use sodium azide with HRP-Conjugated antibodies.
Excessive membrane washing.  Reduce washing step repetitions or duration.
Target protein ran off the gel. Use a positive control and a molecular weight marker matched to the size range of the target protein. 
The target protein is not found in high concentrations in your sample. Maximize the target's concentration by enriching the sample beforehand.
The primary or secondary antibody binds to the blocking agent. Utilize a mild detergent or switch to a different blocking reagent. 
Poor binding of proteins to membrane.  Use a membrane with the correct binding capacity. Dry PVDF membranes after transfer to promote strong binding. 
The protein in the species tested is not recognized by your primary antibody.  Run a positive control. Check literature or perform a BLAST alignment to see whether your antibody should react with the target protein. 
High Background  Insufficient washing or blocking.  Increase blocking time or consider using an alternate blocking reagent. Increase the number of washes. 
Concentration of the primary antibody is too high.  Determine optimal antibody concentration through titration. Use a more dilute antibody with longer incubation times (slow targeted binding is best). 
Secondary antibody binding non-specifically, or binding with blocking agent.  Run a secondary control with no primary antibody. 
Overloaded protein. Decrease the amount of protein loaded on the gel, or dilute the sample.
Contamination of equipment or reagents.  Replace reagents, ensure all equipment is properly cleaned. 
Membrane causing high background. PVDF membranes are considered to give higher background than nitrocellulose membranes. 
Membrane dried out during incubation. Ensure membrane is not drying out during the incubation period. 
Incubation temp too high. Incubate membrane at 4°C.
Cross-reactivity of phospho-specific antibodies with blocking agent. The user may have to try different blocking buffers, such as milk, BSA, etc. to reduce non-specific binding while maintaining specific signals. For optimal results, follow the blocking buffer recommendations from your antibody provider.
Multiple or non-specific bands Primary antibody concentration too high. Decrease the concentration of primary antibody. Run secondary control without the primary antibody. 
Excess protein on gel.  Reduce the amount of protein loaded. 
Issues with blocking.  Optimize blocking time and blocking reagent. 
Insufficient washing.  Increase number of wash steps. 
Antibody not properly purified. Use antibodies purified by the affinity method. 
Target protein has been degraded. Use fresh sample. Include protease inhibitors in your sample buffer. 
Frequently passaged cell lines accumulate differences in protein expression profiles.  Retrieve and expand original cell line, run samples in parallel. 
Target protein has several modified forms (acetylation, methylation, glycosylation etc.). Refer to literature, use agent to remove modifications when possible/necessary. 
Protein subtypes have different molecular weights.  Use bioinformatics analysis and review literature to estimate the correct protein size. 
There are splice variants from the same protein family that share similar epitopes.  Check literature and/or perform a blast search to confirm. 
Multimer formation of target protein. Prior to SDS page, boil protein for 10 min to disrupt multimers. 
Diffuse Bands Concentration of antibody too high. Reduce antibody concentration.
Protein transfer too rapid or gel became over-heated during electrophoresis.  Increase the transfer time and/or run gel at 4°C
Too much protein loaded on gel. Decrease the quantity of protein loaded on gel. 
Smile effect on bands Migration was too rapid. Decrease voltage when running gel. 
Temperature during migration was too high. Run gel at 4°C.
Uneven staining of gel Bacterial contamination of antibodies.  Store antibodies as recommended by your antibody provider. Use fresh buffers. 
Insufficient antibody volume.  Ensure that the membrane is completely covered with antibody and incubate with agitation. 
Target band is extremely high/low on blot Separation during electrophoresis not efficient.  Change gel percentage: use a lower percentage for large proteins, a higher percentage for small proteins. 
Lane with protein ladder is black The antibody is reacting with the protein ladder.  Load gel so there is a blank lane between the ladder and the first sample lane. 
Uneven white spots on blot Air bubbles were trapped between the gel and membrane during transfer. Ensure air bubbles are removed when preparing for transfer.
Black dots on the blot  Antibodies are binding to blocking agent.  Filter blocking agent. 

Direct-Blot™ antibodies are primary antibodies directly conjugated to horseradish peroxidase (HRP), eliminating the need for a secondary antibody. Direct-Blot™ antibodies are able to help you save time and money by eliminating a number of steps and reagents from your normal western blot workflow. This will help you to reduce the cost and to get your experiment completed quicker. Other great benefits of Direct-Blot™ HRP include a shorter protocols, ease of use in multiplexing, and the ability to combine with in Immunoprecipitation assays. In addition, these reagents are highly sensitive and have excellent stability.

 

You can learn more by taking a look at our Direct-Blot™ western blotting protocol and webpage. You can also view our protocol video below to see how it compares to traditional western blotting and learn how it can save you time and money.

Direct-Blot™ HRP anti-β-Actin Antibody

Whole cell lysates (15 µg protein) from HeLa cultures were resolved by electrophoresis (4-20% Trisglycine gel), transferred to nitrocellulose, and probed with 1:10,000, 1:40,000, and 1:100,000 dilutions of Direct-Blot™ HRP anti-β-actin Antibody, clone W16197A, or a 1:10,000 dilution of purified anti-β-actin Antibody, clone W16197A (upper). Proteins were visualized using a 1:3000 diluted HRP anti rat-IgG secondary antibody for purifi ed anti-β-actin Antibody and chemiluminescence detection. Ponceau S staining was used as loading control (lower). Lane M: MW ladder.

 

 

We provide highly specific primary antibodies to detect your proteins of interest, secondary reagents for visualization, and loading control antibodies to help interpret your results. BioLegend also provides molecular weight ladders and ECL substrate kits with the same commitment to quality and value that customers have come to expect from us. To learn more about how BioLegend can help you tame that Wild Wild Western, check out our video on the right!


Featured Products

Antibody Sampler Kits

BioLegend offers a variety of antibody sampler kits that provide an affordable solution for sampling of reagents to further your studies.  These sampler kits include some of the most researched targets BioLegend has to offer, including APP/Aβ, α-Synuclein, HDAC, Tau, and many others.

 

Kit Features:

  • Up to 9 antibodies per kit
  • Antibodies can be used for various applications such as western blot, immunoprecipitation, protein purification, and microscopy
  • 25 μg antibody per vial
  • Sufficient to perform 5-50 mini-gel western blot experiments or immunostaining of 25 slides for IHC, as appropriate
  • Each antibody is quality tested by western blot or IHC in-house
  • Kits are 100% satisfaction guaranteed

View all sampler kits...

 

 

Prime-Step™ Prestained Broad Range Protein Ladder

The Prime-Step™ Prestained Broad Range Protein Ladder is a three-color protein standard with 10 prestained chromophore-conjugated recombinant proteins covering a wide range of molecular weights, from 6.5 to 270 kDa. It is ideal for monitoring protein separation during SDS-PAGE, confirming WB transfer on membranes, and estimating protein sizes.

 

Western-Ready™ ECL Substrate Kit

Western-Ready™ ECL Substrate Kit can be used for enhanced chemiluminescence detection of horseradish peroxidase (HRP) activity from WB reagents.

 

Features include:

  • ECL substrate with high sensitivity
  • A luminol-based detection system
  • Suitable for CCD imager and film
  • Sufficient for detection of 10 mini-blots (Cat. No. 426302) or 100 mini-blots (Cat. No. 426303)
  • Signal duration up to 4 hours
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