How to determine the specificity of an antibody for Western Blot

Paramount for a successful Western blot, is the ability of the primary antibody to detect the protein of interest. But how to validate an antibody for Western blot?

The Western blot application combines the resolving power of polyacrylamide gel electrophoresis, the specificity of antibodies, and the sensitivity of enzyme assays.

Ideally, an antibody only binds to the protein of interest, so only one band should be visible on your gel, and the thickness of the band should correspond to the amount of protein present. Simple, right? Unfortunately, quite often, the antibody catches additional bands below or above the protein of interest. These non-specific proteins overshadow the correct band in intensity thus hampering the quantification.

The choice of primary antibody for the experiment depends on the antigen you want to detect and on how the protein of interest folds, as it exposes different epitopes under different conditions. Both polyclonal and monoclonal antibodies, labeled or unlabeled, work well.

Figure 1. Schematic figure of antibody-based protein detection in WB. Following sample preparation, gel electrophoresis, membrane transfer, and blocking, the primary antibody is applied for the detection of the target proteins. Antibody binding is visualized by chemiluminescence detection in a CCD-camera system using horseradish peroxidase (HRP) labeled secondary antibody. 

Once you have selected your primary antibody, it is important to optimize its concentration by running a calibration curve. This will avoid any subsequent analysis that could cause misleading and confusing interpretations. High primary antibody concentration is a common reason for poor results, such as high background, nonspecific bands, or excessive signal intensity. The primary antibody should be thoroughly assessed and validated to be specific, sensitive, and reproducible enough to detect the intended target protein when used at the lowest concentration in your specific context.

Enhanced antibodies validation for Western Blot: 5 methods

A single band at the expected molecular weight is a great start, but it’s not enough for thorough antibody validation.

Antibody validation is the procedure in which the (primary) antibody is systematically assayed for sensitivity, specificity, and reproducibility. Validation plays a key role in the reproducibility in Western blotting (and other immunoassays), as inconsistent antibody performance leads to great variability. Besides the technical aspects, the poor use of clear, internationally accepted standards for antibody validation and reporting of experimental details contributes to the irreproducibility problem. 

In this paper, Fredrik Edfors, from the Human Protein Atlas (HPA), describes a path forward for systematic validation of antibodies based on the five pillars proposed earlier (Uhlén, M. et al.) here adapted for the WB applications (figure 2). 

Read on, we briefly summarize the five methods for enhanced antibody validation in WB below.

Figure 2. Summary of the five-pillar for WB antibody validation used within the Human Protein Atlas HPA. The validation strategies here described can investigate and validate antibodies that yield several bands in the WB assay. From Edfors F. et al., (2018).

1. Orthogonal validation: protein target confirmed by a non-antibody-based method.

One orthogonal approach is to compare antibody staining intensities to RNA-Seq data from the same samples, over multiple tissues or cells with varying expression of the target protein. The antibody is specific when its signal matches the RNA levels in the tested samples.

Figure 3. Example of the orthogonal antibody validation. Western blot analysis in human cell lines SK-MEL-30 and Caco-2 using Anti-RAB27A antibody. Corresponding RAB27A RNA-seq data for the same cell lines is on the right. Loading control: Anti-HSP90B1.

2. Independent antibody validation: protein target confirmed by an antibody targeting a different epitope of the same protein.

If the two antibodies generate a similar staining pattern when compared in a set of relevant tissues, the antibodies validate each other.

Figure 4.  Example of the independent antibody validation. Western blot analysis using Anti-SUCLG1 antibody HPA036683 (A) shows a similar pattern to independent antibody HPA036684 (B).

3. Genetic validation: antibody specificity confirmed by genetic silencing.

Another test is to perform the same experiments by comparing your sample to a negative sample from a tissue known not to express the intended target of interest or samples derived from genetically ablated cells or animals (siRNA) or knock-out cells/animals (CRISPR): in this case, the antibody should detect the protein only in your sample so, expect to see only one band at the correct molecular weight. An orthogonal method, like qPCR, should ideally verify these results. 

Figure 5.  Example of the genetic antibody validation. Western blot analysis in HEK293 cells transfected with control siRNA, target-specific siRNA probe #1 and #2, using Anti-GLUL antibody.

4. Recombinant expression validation: protein target confirmed by an over-expressed or tagged version of the protein

Begin your experiment by assaying your sample against a positive lysate known to contain the protein of interest or a purified control. In both cases, the antibody should produce a detectable band of the correct molecular weight.

Figure 6.  Example of the recombinant antibody validation. Western blot analysis in control (vector only transfected HEK293T lysate) and GPRC5C over-expression lysate (Co-expressed with a C-terminal Myc-DDK tag (~3.1 kDa) in mammalian HEK293T cells, LY429581).

5. Migration Capture MS validation: the presence of protein target verified by mass spectrometry. 

In this enhanced validation method, the staining pattern and the protein size detected by the same antibody, are compared with results obtained by a capture Mass Spectrometry (MS) method. The specificity of the antibody is confirmed when the size detected by the antibody is equivalent to the size of the corresponding target protein detected in migration capture MS.

The results of the study by Edfors et al. show the value of using different independent validation methods (even without the need for any prior knowledge of the target protein) suggesting that the validation applying one method alone provides enough confidence around the target specificity of the antibody. With this said, additional validation methods further support the specificity of the antibody. 

However, it is important to point out that the enhanced validation is specific for a certain sample context depending on the sample preparation procedures used to test the assay including the relative abundance of the target protein.

Validation data should always be included in the results

In conclusion, fundamental for any successful WB is the consideration of several factors which if not handled appropriately may render data useless.

Although there are no easy solutions for all WB parameters that may fail related to protein size and antibody validity, we firmly believe that researchers ought to include, and reviewers should insist on demanding, fully filed antibody details including catalog number and batch details, within the manuscripts they are reviewing.

Validation data should always be included in the results along with references where the actual antibody described has been used.