ProSci’s Advanced Antibody Validation
High-performance antibodies require a vigorous quality control process that goes above and beyond traditional antibody testing methods. To verify the specificity of antibodies, advanced validation strategies are used, including:
- Genetic modifications (knockout and knockdown)
- Cell Treatment (up or down-regulation)
- Independent Antibody Validation (IAV)
- Relative Expression (compared to known expression profile)
How an antibody performs not only depends upon the samples used but the protocol and assay as well as the antibody itself. ProSci has over twenty years of antibody development and validation experience and uses select techniques in validating its antibodies to ensure antibody specificity, sensitivity, and experimental reproducibility.
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.
Relative Expression (RE)
The basal expression levels of proteins are different in different cell lines and tissues. The specificity of antibodies can be determined based on the natural expression levels of the target protein in multiple cell line or tissue samples. Expression profile information can be confirmed using one of the many online databases such as GeneCards Human Protein Atlas, NCBI and UniProt. Note that protein information is always being updated as scientific knowledge expands.