• Welcome to our NEW website!

Cart 0


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

Correct binding is verified by comparing the band size with the theoretical mass of the target protein. Factors that can affect protein migration, such as alternative isoforms and post-translational modifications are considered, 

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. When suitable cell lines are not available, recombinantly produced full-length target proteins in the form of HEK-293 cell line over-expression lysates are used as positive control samples. The endogenous protein lysates from mouse and rat cell lines are tested for many antibodies,

The peroxidase (HRP) labeled antibody is visualized by chemiluminescence detection using a CCD-camera system.

 

Anti-UCHL1 Antibody
Anti-UCHL1 Antibody

Anti-UCHL1 Antibody

HPA005993
In Stock (10+)
3 490 kr
Anti-ELF2 Antibody
Anti-ELF2 Antibody

Anti-ELF2 Antibody

HPA006057
In Stock (10+)
4 352 kr
Anti-INSL6 Antibody
Anti-INSL6 Antibody

Anti-INSL6 Antibody

HPA021364
In Stock (10+)
4 352 kr
Anti-LRSAM1 Antibody
Anti-LRSAM1 Antibody

Anti-LRSAM1 Antibody

HPA021403
In Stock (10+)
4 352 kr
Anti-DCAF8 Antibody
Anti-DCAF8 Antibody

Anti-DCAF8 Antibody

HPA027218
In Stock (10+)
4 352 kr
Anti-KRT20 Antibody
Anti-KRT20 Antibody

Anti-KRT20 Antibody

HPA027236
In Stock (7)
4 106 kr
Anti-C6orf1 Antibody
Anti-C6orf1 Antibody

Anti-C6orf1 Antibody

HPA037660
In Stock (10+)
4 106 kr
Anti-QRICH1 Antibody
Anti-QRICH1 Antibody

Anti-QRICH1 Antibody

HPA037677
In Stock (10+)
4 106 kr
Anti-SLC13A5 Antibody
Anti-SLC13A5 Antibody

Anti-SLC13A5 Antibody

HPA044343
In Stock (10+)
4 106 kr
Anti-ZNF329 Antibody
Anti-ZNF329 Antibody

Anti-ZNF329 Antibody

HPA044373
In Stock (10+)
4 352 kr
Anti-NUP85 Antibody
Anti-NUP85 Antibody

Anti-NUP85 Antibody

HPA062595
In Stock (10+)
4 352 kr
Anti-COX16 Antibody
Anti-COX16 Antibody

Anti-COX16 Antibody

HPA062659
In Stock (10+)
4 352 kr

 

Enhanced Validation in WB

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 (verifying with a method other than antibodies)
  • Genetic validation (downregulation of the target gene)
  • Independent Antibody Validation (comparing two or more antibodies targeting different regions of the same protein)
  • Recombinant Expression Validation (validating with an over-expressed version of the target protein)

 

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.

 

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.


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).

 

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-P4HA2 Antibody
Anti-P4HA2 Antibody

Anti-P4HA2 Antibody

AMAb90710
In Stock (10+)
4 352 kr
Anti-MST1R Antibody

Anti-MST1R Antibody

AMAb90766
In Stock (10+)
4 106 kr
Anti-STAT3 Antibody
Anti-STAT3 Antibody

Anti-STAT3 Antibody

AMAb90776
On demand
3 490 kr
Anti-BRD4 Antibody
Anti-BRD4 Antibody

Anti-BRD4 Antibody

AMAb90841
In Stock (10+)
4 106 kr
Anti-PARP1 Antibody
Anti-PARP1 Antibody

Anti-PARP1 Antibody

AMAb90959
In Stock (10+)
3 490 kr
Anti-HNRNPC Antibody
Anti-HNRNPC Antibody

Anti-HNRNPC Antibody

AMAb91010
In Stock (10+)
4 106 kr
Anti-CNP Antibody
Anti-CNP Antibody

Anti-CNP Antibody

AMAb91068
In Stock (10+)
4 106 kr
Anti-GAPDH Antibody
Anti-GAPDH Antibody

Anti-GAPDH Antibody

AMAb91152
In Stock (10+)
3 490 kr
Anti-TLE3 Antibody
Anti-TLE3 Antibody

Anti-TLE3 Antibody

AMAb91188
In Stock (10+)
4 352 kr
Anti-CDH1 Antibody
Anti-CDH1 Antibody

Anti-CDH1 Antibody

AMAb90865
In Stock (10+)
3 490 kr
Anti-FAM122B Antibody
Anti-FAM122B Antibody

Anti-FAM122B Antibody

HPA000266
In Stock (10+)
4 352 kr
Anti-ACAA1 Antibody
Anti-ACAA1 Antibody

Anti-ACAA1 Antibody

AMAb91020
In Stock (10+)
4 352 kr