Advanced Validation Methods
Knockout refers to samples where the genetic code for a particular protein is completely removed, resulting in samples that do not express the protein of interest. Therefore, KO tissues or cell lines function as true negative controls and are used in parallel with wild type samples to validate antibodies and show they are specific for their target with a positive signal in the wild type, non-knockout sample, and a negative signal in the KO sample. KO cell lines are generated through genetic tools such as CRISPR. Knockout samples can be used in a wide variety of assays, including western blot, IHC, ICC, and flow cytometry.
Knockdown uses siRNA to knock down the gene of interest, which reduces the expression level of the target protein. It is still a useful negative control when run in parallel with wild type (WT) samples by confirming downregulation of the target of interest with specific siRNA. Antibodies specific to the target of interest would not show as strong of a signal in the KD samples as with the WT samples. Knockdown samples can be used in a wide variety of assays, including western blot, IHC, ICC, and flow cytometry.
Cell Treatment (CT)
Utilizing known stimuli that affect the target protein expression, cell treatment is another useful tool for antibody validation. Cell culture conditions are modified or chemical introduced to either decrease or increase protein expression levels. Therefore, cell treatment, depending upon the conditions, can create either positive or negative controls in multiple assays, including western blot, IHC, ICC, and flow cytometry.
Independent Antibody Validation (IAV)
Independent Antibody Validation uses two or more antibodies against different epitopes of the target protein in multiple tissue or cell line samples to compare their respective expression profiles. Specificity of the antibodies is gauged based upon the similarity of the expression profiles; if they are similar or identical, the antibodies are likely detecting the correct protein, but if they are different then the specificity of at least one of the antibodies is in question and further validation is required.