A guide to antibody validation

The most commonly used methods to validate antibody specificity.

To ensure accurate and consistent results, it’s important you take the time to ensure that the antibody performs as expected in your experimental setup. A good supplier will have already tested the antibody, but it’s impossible to account for the huge number of different protocols and reagents that the antibody may be used with once in the hands of the researcher. Your own validation steps are essential because they are specific to your setup.




KO models


Protein-encoding gene eliminated with genetic tools (eg CRISPR)

KO cell lines function as a true negative control

Guaranteed no expression of the target gene

A potentially large number of KO cell lines may be generated in a short period of time

Knockout cell lines may be used in all assays - western blot, IHC, ICC, flow cytometry


Knockout cell lines against specific genes are not always viable

Is a very powerful negative control, not a confirmation of binding

Mass spectrometry/ IP-MS


Protein complexes characterized by first immunoprecipitating them from a cell lysate and then analyzing with mass spectrometry

Amenable to a high-throughput format

Potential to estimate abundance of target protein bound to the antibody of interest using normalization techniques

Can recognize all isoforms

Can identify PTMs

Can identify interacting partners and complexes

Can identify off-targets

Confirms specificity based upon digested protein fragments

Potential to estimate abundance of target protein bound to the antibody of interest using normalization techniques

IP-MS assays difficult to optimize

Too many washes of your IP could remove weak or moderate binders

Not all antibodies are suitable for IP

For some targets, protein binding to the antibody of interest via IP is ineffective

Can be difficult to distinguish partner proteins pulled down in a complex from off-target binding

Interpreting the data can be tricky as the highest enrichment score does not always mean that this is the target the antibody preferentially binds to, for example:

1) Large number of interactors found with higher fold enrichment: If your protein of interest is in high abundance in your sample but its interacting partners are not, then the fold enrichment for the interacting partners could be higher than for your protein of interest if it is pulling multiple units of your interacting partner

2) Antibody detecting multiple family members: if one is present at a lower level in the starting lysate, then the fold enrichment is likely to be higher


Western blot


Protein detected and quantified in a sample via initial separation by size and then blotting onto a membrane to be detected by an antibody

Useful for determining antibody specificity against target protein based upon molecular weight

Ideal for detecting either native or denatured proteins

Qualitative assay

Time-consuming assay

Difficult to determine the optimal experimental conditions (ie methodology and buffer)

Only a small number of antibodies may be validated per run



Protein in tissues (IHC) or cells (ICC) detected via specific antibodies and reporter molecules

Validates whether an antibody recognizes the correct protein based on cellular localization

Specificity confirmed based on cells that either do or do not express the target protein

Qualitative assay

Unable to determine if an antibody recognizes other proteins non-specifically with identical cellular localization

Often difficult to determine cell or tissue types that either do or do not target the protein

Protein/peptide array


Antibody binding events detects by first spotting arrays are spotted with the peptides/protein and then adding the antibody (similar to ELISA)

Allows screening of a larger number of over-expressed proteins

Very high-throughput screening process

Requires very small sample volumes

Protein array only: unable to screen for post-translationally modified proteins 

Only present linear epitopes for interrogation – do not usually present conformational epitopes

Do not usually present conformational epitopes

siRNA knockdown


Protein-encoding gene expression is lowered with genetic tools (eg interfering RNA)

Confirms specificity through target protein being downregulated

Knockdown cells lines may be used in all assays – western blot, IHC, ICC, flow cytometry

Knockdown is transient

Difficult experiment to optimize – often requiring several siRNA sequences

The KD could be ineffective so good controls are needed eg use of multiple RNAs targeting the same gene

Non-specific reduction in expression might be observed where the siRNA binds and silences “off-targets” due to binding to similar transcripts

Expression profiles (online)


Expected protein expression is checked via online databases

Quick and easy to run without the need for any equipment

Large number of online databases available, for example

1) www.genecards.org

2)  portals.broadinstitute.org/ccle

3) www.ncbi.nlm.nih.gov/gene

4) www.proteinatlas.org


Expression data may not be complete in some cases

Can be difficult to find negative cell lines or tissues

Cell treatment


Protein expression level manipulated within cells

Relatively straightforward to increase or reduce expression levels of certain proteins (eg via starvation or chemically)

These could serve as positive or negative controls


Phosphatase treatment: tells you it is phospho-specific but not to which site

You need to have additional controls in place to ensure the cell treatment worked

Other common methods do not test antibody specificity, and we do not recommend you use them alone. These include

  • Blocking with immunizing peptide: even non-specific antibodies will be blocked by immunizing peptide
  • Omission of primary: can be used to evaluable the tissue or secondary detection reagents but not the primary antibody

Additional points to consider when validating an antibody

1. Choice and preparation of cell lines or tissue samples – true positive and negative controls are essential

It is important to choose samples that express the target protein of interest for positive controls, as well as cell lines that do not express the protein as a true negative control.

Such information may be sourced from peer-reviewed papers and by using online protein databases. Furthermore, antibodies may or may not recognize the protein in its native or denatured state. It is therefore important to prepare the test samples accordingly. For example, an antibody that recognizes the protein only in its native form should not be used on samples using denaturing conditions, for instance in western blotting.

See our sample preparation guides for western blot or ELISA.

2. Protocols

Ensure an optimized protocol is used to give the antibody the best chance of passing the validation process. For instance, incubation times can vary dramatically from a minimum of one hour to overnight at 4°C, so the optimal incubation period for each antibody will have to be determined. If the period is too short, sensitivity issues may be encountered, but incubation for too long may lead to background staining. Other factors – such as working dilutions, blocking conditions and the use of native vs denatured conditions – will also need to be individually optimized.

See our antibody protocols and troubleshooting tip guide.

3. The choice of buffers

The majority of antibody assays will use two buffer types: either PBS or TBS. The optimal buffer will need to be determined. Additionally, subtleties such as pH should also be taken into consideration.

See our buffer recommendations for optimal western blot results and antibody storage.

Also link to the following pages:

•    KO validation overview page
•    http://www.abcam.com/content/validating-our-products
•    http://www.abcam.com/index.html?pageconfig=popular_protocols
•    http://www.abcam.com/tag/antibody%20guide
•    Scientists helping scientists- http://www.abcam.com/index.html?pageconfig=support
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