How can I determine if a protein has been knocked out?

Posted April 24, 2020

Answer

To determine is a protein has been knocked out, one of the best options is to conduct a western blot (also known as an immunoblot). By employing an antibody specific for the protein of interest, a western blot can allow you to compare the knockout clone with the wild-type and determine if a protein has been successful knocked out. Another method for screening if a knockout is successful is RT-qPCR, which can allow you to analyze gene expression.

Additional resources

Bara, A.M., Messana, A., Herring, A., Hazelbaker, D.Z., Eggan, K., Barrett, L.E. (2016). Generation of a TLE3 heterozygous knockout human embryonic stem cell line using CRISPR-Cas9. Stem Cell Research, 17(2), 441-443. doi:10.1016/j.scr.2016.09.008

Estep, J. A., Sternburg, E. L., Sanchez, G. A., & Karginov, F. V. (2016). Immunoblot screening of CRISPR/Cas9-mediated gene knockouts without selection. BMC molecular biology, 17, 9. doi:10.1186/s12867-016-0061-0