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Western Blot (WB)

Learn how our antibodies are validated in Western blot, and find the protocols you need to be successful in your experiment.

Western blot detects proteins separated on a gel by their size and targeted with specific antibodies. The protein separation is performed by gel electrophoresis. 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.

Western blot Standard Protocol
Western blot Protocol - BSA Blocking

Validation in Western Blot

Our Triple A Polyclonals and PrecisA Monoclonals are routinely validated in Western blot to ensure correct binding. We verify the band size against the theoretical mass of the target protein, considering factors like isoforms and post-translational modifications.
Endogenous protein lysates from human tissues and cell lines are primarily used for validation. When suitable lysates are not available, recombinant full-length target proteins (e.g., from HEK-293 over-expression systems) are used as positive controls. Mouse and rat cell lysates are also tested for many antibodies. The peroxidase-labeled antibody is detected using chemiluminescence and a CCD camera system.

Enhanced Validation in Western Blot


To further confirm antibody specificity, we apply four enhanced validation methods:

  1. Orthogonal Validation – Verifying with a non-antibody-based method, such as RNA-Seq.
  2. Genetic Validation – Down-regulation of the target gene.
  3. Independent Antibody Validation – Comparing antibodies targeting different regions of the same protein.
  4. Recombinant Expression Validation – Validating with an over-expressed version of the target protein.

 

1. Example of orthogonal validation by comparing antibody signal in WB to RNA

The orthogonal validation method validates the antibody staining using a non-antibody-based method.

For example, the Western blot results are compared with RNA-Seq data for the same samples, using both positive and negative controls. 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 validation: one with high RNA expression and the other with low or no RNA expression of the target protein. The cell lines are selected to express 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. 

 

Example of orthogonal validation in WB, 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.

 

2. Example of genetic validation in WB 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 the corresponding gene's knockdown levels correlate 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 in WB 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.


3. Example of validation by independent antibody in WB

Validation by independent antibody 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. 

Example of independent antibody validation in WB using the Anti-PBLD antibody HPA038036 (A) shows a similar pattern to the independent antibody HPA038035 (B).

 

4. Example of recombinant expression validation in WB

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 line. 

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

Example of recombinant expression validation in WB 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).

 
The importance of loading controls in WB

Loading controls are helpful to ensure that an even amount of protein is loaded on the gel. 

When studying the expression of a target protein in different samples, it is necessary to normalize protein levels using a loading control antibody 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.

Suitable targets for loading control are housekeeping proteins expressed at high levels in a wide range of cell lines and tissues. 

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 for WB

When choosing a loading control, 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 WB

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 host species than the antibody targeting the protein of interest.

Using suitable secondary antibodies conjugated with fluorescent dyes, the western blot protocol can be reduced to only one primary and one secondary incubation step. The two bands can be visualized in the same image.  

 
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. The Atlas Antibodies Loading Control panel contains antibodies of different isotypes allowing multiplexing. 

Browse loading control antibodies

 

Anti-CNOT3 Antibody
Anti-CNOT3 Antibody

Anti-CNOT3 Antibody

HPA006408
In Stock (7)
4 570,0 kr
Anti-ZSCAN22 Antibody
Anti-ZSCAN22 Antibody

Anti-ZSCAN22 Antibody

HPA006471
In Stock (10+)
4 570,0 kr
Anti-TMTC2 Antibody
Anti-TMTC2 Antibody

Anti-TMTC2 Antibody

HPA026954
In Stock (10+)
4 570,0 kr
Anti-PPP1R3C Antibody
Anti-PPP1R3C Antibody

Anti-PPP1R3C Antibody

HPA027041
In Stock (10+)
4 570,0 kr
Anti-STYX Antibody
Anti-STYX Antibody

Anti-STYX Antibody

HPA040290
In Stock (10+)
4 570,0 kr
Anti-CEP57L1 Antibody
Anti-CEP57L1 Antibody

Anti-CEP57L1 Antibody

HPA040378
In Stock (10+)
4 570,0 kr
Anti-CFAP45 Antibody
Anti-CFAP45 Antibody

Anti-CFAP45 Antibody

HPA043618
In Stock (10+)
4 570,0 kr
Anti-CCDC94 Antibody
Anti-CCDC94 Antibody

Anti-CCDC94 Antibody

HPA055675
In Stock (10+)
4 311,0 kr
Anti-AP5M1 Antibody
Anti-AP5M1 Antibody

Anti-AP5M1 Antibody

HPA055768
In Stock (5)
4 570,0 kr
Anti-PPP4R3A Antibody
Anti-PPP4R3A Antibody

Anti-PPP4R3A Antibody

HPA063917
In Stock (10+)
4 570,0 kr
Anti-TM9SF4 Antibody
Anti-TM9SF4 Antibody

Anti-TM9SF4 Antibody

HPA064099
In Stock (10+)
4 570,0 kr
Anti-CAV2 Antibody
Anti-CAV2 Antibody

Anti-CAV2 Antibody

HPA044810
In Stock (10+)
4 311,0 kr
Anti-SUN3 Antibody
Anti-SUN3 Antibody

Anti-SUN3 Antibody

HPA008344
In Stock (10+)
4 570,0 kr
Anti-FCER2 Antibody
Anti-FCER2 Antibody

Anti-FCER2 Antibody

HPA008928
In Stock (10+)
4 311,0 kr
Anti-SFRP4 Antibody
Anti-SFRP4 Antibody

Anti-SFRP4 Antibody

HPA009712
In Stock (10+)
4 570,0 kr
Anti-NT5C3A Antibody
Anti-NT5C3A Antibody

Anti-NT5C3A Antibody

HPA010630
In Stock (10+)
4 570,0 kr
Anti-SPATA9 Antibody
Anti-SPATA9 Antibody

Anti-SPATA9 Antibody

HPA010848
In Stock (10+)
4 570,0 kr
Anti-SMIM19 Antibody
Anti-SMIM19 Antibody

Anti-SMIM19 Antibody

HPA011145
In Stock (10+)
4 570,0 kr
Anti-SEC23IP Antibody
Anti-SEC23IP Antibody

Anti-SEC23IP Antibody

HPA043305
In Stock (10+)
4 570,0 kr
Anti-RACGAP1 Antibody
Anti-RACGAP1 Antibody

Anti-RACGAP1 Antibody

HPA043912
In Stock (10+)
4 311,0 kr
Anti-RPL8 Antibody
Anti-RPL8 Antibody

Anti-RPL8 Antibody

HPA045095
In Stock (10+)
4 570,0 kr
Anti-SCAF11 Antibody
Anti-SCAF11 Antibody

Anti-SCAF11 Antibody

HPA045456
In Stock (10+)
4 570,0 kr
Anti-ERBIN Antibody
Anti-ERBIN Antibody

Anti-ERBIN Antibody

HPA048606
In Stock (10+)
4 570,0 kr
Anti-ANLN Antibody
Anti-ANLN Antibody

Anti-ANLN Antibody

HPA050556
In Stock (10+)
4 570,0 kr