Quantitative Western Blotting: How and why you should validate your antibodies

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Validating your antibodies is the process of confirming an antibody recognizes your protein of interest with low cross-reactivity to other targets. Validating antibodies is critical for ensuring consistent, reproducible results for Western blotting. It is frequently required before submission to a journal, is a step that is sometimes overlooked and skipped. We might even assume because we validated the antibody for immunopreciptation, we don’t need to validate it forWestern blotting. However, this is far from the truth! Binding is highly dependent on experimental conditions, which is why it’s important to validate antibodies in similar conditions to those in your protocol.

Quantitative Western blotting in particular depends on confirming the signal you’re getting is coming only from your protein of interest. Without antibody validation (and validation that the signal is linearly related to the amount of protein), you won’t have the accuracy and precision you need for quantitative analysis. After all, the goal of the study is to measure the abundance of a specific protein, so cross-reactivity can artificially inflate your abundance measurement.

To ensure experimental quality, the International Working Group for Antibody Validation gives five different methods for validating antibodies, four of which work for Western Blotting. The Group recommends using at least two of the validating strategies we will discuss below.

Genetic Strategies

The genetic strategies approach should be familiar to every biochemist who’s been in the game for a while, though the methods are constantly being updated. Measure the relevant signal in control cells or tissues in which the target epitope has been knocked out or knocked down using CRISPR-Cas9 or RNA interference (RNAi). Because there should be very little to no expression of the target protein, any signal observed will indicate crossreactivity.

Western blot of protein lysate from a cell line with a particular protein knocked down by two different siRNA molecules

For example: In this Western blot, we see protein lysate from a cell line with a particular protein knocked down by two different siRNA molecules in columns 1 and 2. Column C is control. It’s possible to determine how much off-target binding there will be by comparing the columns.

Orthogonal Strategies

This next antibody validation method is a little more low-tech. Use an antibody-independent method to quantify the target signal for several samples. Then compare these amounts with those from antibody-based approaches. There are several targeted proteomics approaches that can quantify protein expression in different samples. Antibody labeling should correlate with these quantifications.

comparison quantification of protein expression in eight different cell lines using western blot and mass spectrometry
This figure compares quantification of protein expression in eight different cell lines using western blot and mass spectrometry. With a sufficiently high correlation, we can conclude that the antibody is valid for Western blot analysis.

Independent antibody strategies

Using two or more different antibodies against the same target is an intuitive and effective method to gauge the specificity of an antibody. It’s important to test the correlation within one application environment. You’ll know if the antibodies are independent if they have different epitopes (bind to different regions of the protein). Having different epitopes means they are unlikely to exhibit off-target binding to the same protein that’s unrelated to the study. The independent antibody smethod pairs well with a knockout or knockdown approach.

Western blot using Independent antibody strategy where two independent antibodies are used against a protein
This figure shows two independent antibodies used against a protein. We can determine the correlation of the signals from each of the eight cell lines. Be sure to check the entire gel because bands indicative of off-target binding may be elsewhere. 

Expression of tagged proteins

It is also possible to validate antibodies by expressing a protein that has an affinity tag (e.g. FLAG or v5) or a fluorescent protein (such as GFP or YFP). It is then possible to match the antibody expression with that of the other method. This approach is limited because it’s best to tag the endogenous gene. Overexpressing the target protein might artificially drown out off-target binding, meaning the method won’t be effective for validation.

We hope this article was helpful and you will use it as a reference piece for your next experiment. As a reminder: you should always include validation data in your results. 

Products to help confirm your antibody is binding specifically and selectively:

Vocab used in this blog post
AffinityBond strength between the antibody’s paratope and the antigen’s epitope
AntibodyImmunoglobulin protein that recognizes a specific antigen or target of interest
Cross-reactivity/non-specific bindingBinding of the antibody to other sample proteins with epitopes similar to the target protein
SpecificityRecognition of the target antigen by the antibody
SelectivityPreferential binding of the antibody to the target antigen in a complex sample
quantitative western blot basics


Get a quick overview of the steps you can take to ensure your Western blots are quantitative. This free guide also includes a troubleshooting section and tear-out quantitative Western blotting checklist.

  1. Uhlen, et al. A proposal for validation of antibodies. Nature Methods 13(10), 2016, 823-827

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