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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-PTPN2 Antibody
Anti-PTPN2 Antibody

Anti-PTPN2 Antibody

HPA015004
In Stock (10+)
4 311,0 kr
Anti-STK10 Antibody
Anti-STK10 Antibody

Anti-STK10 Antibody

HPA015083
In Stock (10+)
4 570,0 kr
Anti-LYRM4 Antibody
Anti-LYRM4 Antibody

Anti-LYRM4 Antibody

HPA030362
In Stock (10+)
4 570,0 kr
Anti-TXLNB Antibody
Anti-TXLNB Antibody

Anti-TXLNB Antibody

HPA030403
In Stock (10+)
4 570,0 kr
Anti-PSMG2 Antibody
Anti-PSMG2 Antibody

Anti-PSMG2 Antibody

HPA041517
In Stock (10+)
4 570,0 kr
Anti-KARS Antibody
Anti-KARS Antibody

Anti-KARS Antibody

HPA041550
In Stock (6)
4 570,0 kr
Anti-TNS3 Antibody
Anti-TNS3 Antibody

Anti-TNS3 Antibody

HPA055338
In Stock (10+)
4 311,0 kr
Anti-HNRNPA0 Antibody
Anti-HNRNPA0 Antibody

Anti-HNRNPA0 Antibody

HPA059404
In Stock (10+)
4 570,0 kr
Anti-DDX3X Antibody
Anti-DDX3X Antibody

Anti-DDX3X Antibody

HPA059585
In Stock (10+)
4 311,0 kr
Anti-NR4A1 Antibody
Anti-NR4A1 Antibody

Anti-NR4A1 Antibody

HPA059742
In Stock (10+)
4 311,0 kr
Anti-HNRNPA2B1 Antibody
Anti-HNRNPA2B1 Antibody

Anti-HNRNPA2B1 Antibody

HPA065537
In Stock (10+)
4 311,0 kr
Anti-QDPR Antibody
Anti-QDPR Antibody

Anti-QDPR Antibody

HPA065649
In Stock (10+)
4 570,0 kr
Anti-TRMT1L Antibody
Anti-TRMT1L Antibody

Anti-TRMT1L Antibody

HPA026728
In Stock (10+)
4 570,0 kr
Anti-SFXN2 Antibody
Anti-SFXN2 Antibody

Anti-SFXN2 Antibody

HPA026834
In Stock (10+)
4 570,0 kr
Anti-TSEN2 Antibody
Anti-TSEN2 Antibody

Anti-TSEN2 Antibody

HPA027125
In Stock (10+)
4 570,0 kr
Anti-ACTL8 Antibody
Anti-ACTL8 Antibody

Anti-ACTL8 Antibody

HPA027176
In Stock (10+)
4 570,0 kr
Anti-VEGFD Antibody
Anti-VEGFD Antibody

Anti-VEGFD Antibody

HPA027342
In Stock (10+)
4 311,0 kr
Anti-MMACHC Antibody
Anti-MMACHC Antibody

Anti-MMACHC Antibody

HPA027399
In Stock (10+)
4 570,0 kr
Anti-RPS6KL1 Antibody
Anti-RPS6KL1 Antibody

Anti-RPS6KL1 Antibody

HPA027451
In Stock (10+)
4 570,0 kr
Anti-IDO1 Antibody
Anti-IDO1 Antibody

Anti-IDO1 Antibody

HPA027772
In Stock (10+)
3 665,0 kr
Anti-DMRT1 Antibody
Anti-DMRT1 Antibody

Anti-DMRT1 Antibody

HPA027850
In Stock (10+)
4 570,0 kr
Anti-WDTC1 Antibody
Anti-WDTC1 Antibody

Anti-WDTC1 Antibody

HPA028177
In Stock (10+)
4 570,0 kr
Anti-YOD1 Antibody
Anti-YOD1 Antibody

Anti-YOD1 Antibody

HPA028400
In Stock (10+)
4 570,0 kr
Anti-DIRAS3 Antibody
Anti-DIRAS3 Antibody

Anti-DIRAS3 Antibody

HPA028483
In Stock (10+)
4 570,0 kr