Western Blot

Learn how our antibodies are validated in Western blot, and how to be successful in your Western blot experiment.

Western blot an application used to detect proteins separated on a gel by their size using antibodies specific to the target protein. The protein separation is performed by gel electrophoresis and the proteins are then transferred to a nitrocellulose or PVDF (polyvinylidene fluoride) membrane on which the blotting with the antibody is performed.

Protocols and guides

Use these recommended protocols for optimal results in Western blot using our antibodies. The protocols are optimized for Triple A Polyclonals and PrecisA Monoclonals.

Looking for a different protocol? See all our protocols for IHC, WB and ICC.

Go to protocols

Validation in Western blot

Our antibodies, Triple A Polyclonals, and PrecisA Monoclonals are routinely validated in Western blot. 

Correct target binding is verified by comparing the band size with the theoretic mass of the target protein. Factors which can affect protein migration are taken into consideration, such as alternative isoforms and post-translational modifications.

Endogenous protein lysates from human tissues and cell lines are primarily used as samples. Protein lysates are selected based on RNA expression levels and the scientific relevance of the target. In some cases, suitable cell lines are not available. In these situations, recombinantly produced full-length target proteins in the form of HEK-293 cell line over-expression lysates are used as positive control samples. For a large number of antibodies, endogenous protein lysates from mouse and rat cell lines are tested. 

The binding of the antibody is visualized by chemiluminescence detection in a CCD-camera system using a peroxidase (HRP) labeled secondary antibody. 

Enhanced validation in Western blot

To further demonstrate specificity, the validation performed for our antibodies is expanded with application-specific Enhanced Validation. In Western blot, four different enhanced validation methods are applied:

Orthogonal validation by comparing antibody Signal in Western blot to RNA

Orthogonal validation is an enhanced method for validation where the antibody results are confirmed by a non-antibody based method.

The method is applied in Western blot by comparing the Western blot result with RNA-Seq data for the same samples, using both positive and negative samples. Antibody specificity is confirmed when the antibody signal matches RNA levels in the evaluated samples.

For each antibody, two tissues or endogenous cell lines are chosen for the validation, one with high RNA expression and the other with low or no RNA expression of the target. The cell lines are chosen so that there is at least a five-fold difference between the RNA expression in the high and low samples.

In the enhanced validation data presented for the antibodies, the Western blot lanes in the high and low cell lines are displayed together with their corresponding RNA values. 

Orthogonal validation in WB
The image shows an example of orthogonal validation, using the Anti-RAB27A antibody (HPA001333) in Western blot in the SK-MEL-30 and CACO-2 cell lines. On the right-hand side, bars representing the TPM values for RAB27A in the same cell lines are presented.

Genetic validation in Western blot by siRNA knockdown 

Genetic validation by siRNA knockdown is an enhanced method for validation where the target gene is downregulated. Antibody specificity is confirmed when knockdown of the corresponding gene correlates with a decrease in the antibody signal. 

At Atlas Antibodies, two separate siRNA probes are employed to silence each target and a loading control is added to ensure even loading and equal transfer over the gel. The knockdown is approved if at least 50% silencing is achieved for at least one of the two siRNA probes.

In the enhanced validation data presented for the antibody, the Western blot lanes in the control and knocked down samples are displayed together with the loading control, and the relative remaining intensity after silencing is presented. 

Example of genetic validation by siRNA knockdown
Example of genetic validation by siRNA knockdown in Western blot using the Anti-PPIB antibody (HPA012720). U-251 cells have been transfected with control siRNA and two target specific siRNA probes. Downregulation of antibody signal confirms target specificity. The remaining intensity relative control lane is indicated as a percentage.

Validation by independent antibodies in Western blot

Validation by Independent Antibodies is an enhanced method for validation where the antibody specificity is demonstrated by comparing at least two antibodies targeting the same protein with non-overlapping epitopes. 

If the signals from the two antibodies correlate when compared across multiple samples, the antibodies validate each other.

In the validation data presented for the antibody, the Western blots from both antibodies are displayed together.

Validation by independent antibodies in WB
Western blot analysis using Anti-PBLD antibody HPA038036 (A) shows a similar pattern to independent antibody HPA038035 (B).

Recombinant expression validation in Western blot

Recombinant expression validation is an enhanced method for validation where the antibody binding is confirmed using an over-expressed version of the target protein. 

The method is applied to Western blot by comparing the antibody signal in a sample where the target protein has been recombinantly over-expressed, with the signal from a control sample. Antibody specificity is confirmed when the antibody shows a strong band in the cell line with recombinant expression and no or faint band in the control. 

In the validation data presented for the antibody, the Western blot includes the over-expressed sample and the control sample in the same blot.

Recombinant expression validation in WB
Example of recombinant expression validation in Western blot using the Anti-ACY3 antibody (HPA039219). Lane 1: marker, lane 2: negative control (vector only transfected HEK293T lysate), lane 3: ACY3 over-expression lysate (Co-expressed with a C-terminal myc-DDK tag (~3.1 kDa) in mammalian HEK293T cells, LY408962).

See all antibodies validated for Western blot.

Browse antibodies

The importance of loading controls

Loading control antibodies are useful to ensure that an even amount of protein is loaded on the gel. Suitable targets to use as loading control markers are usually housekeeping proteins expressed at high levels in a wide range of cell lines and tissues. 

When studying the expression of a target protein in different samples, it is necessary to normalize protein levels using a loading control antibody in order to get reliable data. By using a loading control you can distinguish an unevenly loaded sample from an actual difference in the protein expression between the samples.

Loading controls can also be used to confirm that the transfer of protein from the gel is equal over the whole membrane. This is particularly useful when comparing protein expressions over several samples.

How to choose a loading control

When choosing a loading control antibody, be sure to pick one that targets a protein of a different molecular weight than that of your protein of interest. By doing so you can easily differentiate between the two bands during the blotting. For optimal results, also check that the protein or RNA expression level of the loading control target is fairly high in the cell lines or tissue lysates you will use.

Save time with fluorescence-based multiplex Western blot

Fluorescence-based multiplex Western blot is a time-saving alternative to chemiluminescent detection. For multiplexing, choose a loading control antibody with a different isotype or different host species than the antibody targeting the protein of interest. Using suitable secondary antibodies conjugated to fluorescent dyes, the western blot protocol can then be reduced to only one primary and one secondary incubation step and the two bands can be visualized in the same image. The Atlas Antibodies Loading Control panel contains antibodies of different isotypes allowing multiplexing. 

Loading controls

Discover our antibody panel of Loading Controls,  consisting of PrecisA Monoclonals targeting proteins that are constitutively expressed in a large set of cell lines and tissues.

Browse loading control antibodies