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Updated May 17, 2022
Antibody validation revolves around proving three key aspects:
Here we will focus on how you can validate antibody specificity in your experimental setup to ensure accurate and consistent results. Although a good manufacturer usually tests an antibody in several applications and species, it's impossible to account for numerous protocols and reagents with which researchers may use the antibody. Therefore, your antibody validation steps are essential because they are specific to your setup.
1. Choice and preparation of positive and negative controls
Identifying and using appropriate positive and negative controls is essential for successful antibody validation.
It's often challenging to determine cell or tissue types that do or do not express the target protein. You can find information about target protein expression in different tissues or cell lines in peer-reviewed papers and online protein databases, including:
However, there are a few limitations to defining protein expression profiles using online databases:
Furthermore, antibodies may or may not recognize the protein in its native or denatured state. It is therefore essential 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, such as western blot.
Ensure you use an optimized protocol 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 you'll need to determine the optimal incubation period for each antibody. If the incubation period is too short, you may encounter sensitivity issues, while prolonged incubation time may lead to background staining. You will also need to optimize other factors, such as working dilutions, blocking conditions, and the use of native vs denatured conditions.
3. The choice of buffers
The majority of antibody assays will use two buffer types: PBS or TBS. You will need to determine the optimal buffer for your experiment, considering parameters that can influence buffer performance, such as pH.
Cell lines or tissues that endogenously express or lack the target protein can serve as positive or negative controls, respectively. You can use several various cell lines with different protein expression levels to provide a range of controls. Alternatively, appropriate positive and negative controls can be designed using multiple methods, including knock-out models, siRNA knockdown and cell treatment (Table 1).
Table 1. Models for designing appropriate positive and negative controls.
Knock-out (KO) models
Cell lines, tissues, or lysates, where the protein-encoding gene of interest is eliminated with genetic tools (eg, CRISPR)
KO models function as a true negative control
Guaranteed no expression of the target gene
You can use KO cell lines or tissues in all assays: western blot, IHC, ICC, flow cytometry
KO cell lines against 'essential' genes are not always viable
The lack of signal in a KO sample shows that the antibody detects the protein of interest in the wild-type sample. It does not guarantee that the Ab will not bind unspecifically to an unrelated protein in a different sample background.
Protein-encoding gene expression is lowered using post-transcriptional gene regulation tools, such as small interfering RNA (siRNA).
Confirms specificity through target protein being downregulated
Knockdown cells lines may be used in all assays: western blot, ICC, flow cytometry
Knockdown is transient
Knockdown is rarely 100% effective, so good controls are needed, such as real-time PCR and well-established siRNA for a control gene.
Non-specific reduction in expression might be observed where the siRNA binds and silences "off-targets" due to binding to similar transcripts
Protein expression level is manipulated within cells
Can increase or reduce expression levels of specific proteins or affect post-translational modifications, such as phosphorylation, (eg, via starvation)
These could serve as positive or negative controls
Additional controls are required to ensure the cell treatment worked
It can be challenging to design the experiment
You can use several different methods to validate antibodies. Below we outline some popular applications, their benefits and their limitations.
Potential to estimate abundance of target protein bound to the antibody of interest using normalization techniques
Can recognize all protein isoforms to which the antibody binds
Can identify post-translational modifications, interacting partners and complexes,
Confirms specificity based upon digested protein fragments
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, off-target binding can be difficult to demonstrate. An isotype control Ab is required, but there may still be discrepancies between IPs.
Protein is detected in a sample via initial size separation and then blotting onto a membrane to be visualized by an antibody
Not easily automatable compared to some of the other applications
Note that the two following methods can't be considered exhaustive tests of antibody specificity; therefore, we do not recommend using them alone: