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Join Dr Hanna Dreja, Abcam’s Principal Quality Control Scientist, as she discusses how knockout cell lines are being used to increase validation standards and support the reproducibility of research across the life science industry.
CRISPR KO is performed by the insertion or deletion of nucleotides in the target gene sequence. This results in a frameshift in the gene sequence that prevents expression of the complete protein as the translational machinery will hit a premature stop codon. As a result, you end up with a tiny protein that will be degraded.
The resulting mRNA undergoes nonsense-mediated decay, as the cell detects that it is not fully translated. However, this process is not 100% efficient 1.
If you have confidence in your Abs, I would recommend using protein detection to determine if your protein is truly knocked out.
To demonstrate that an antibody binds specifically to the protein of interest, one needs to use a negative control – a control sample that does NOT contain this protein. If you have a cell line that normally expresses the protein, and you genetically modify this to no longer express the protein, you have two perfectly matched samples: a positive and a negative control sample. If your antibody is specific, you will no longer detect the signal in the knockout sample.