I transfected SARS-CoV-2 Spike (https://www.addgene.org/141382/) in HEk293T cells and 48 h later collected cell lysate in Pierce lysis buffer containing protease inhibitor cocktail. However, upon running them in reducing SDS PAGE, I am getting faint band at 180kDa and prominent bands at 100 and 70kDa. What could be the reason? I was expecting a clear band at 180kDa corresponding to the full length protein.
Laemmli bufffer with beta mercaptoethanol was used for loading in SDs PAGE.
Hello Rohan, the following publication may help answer your question: https://www.nature.com/articles/s41564-021-00908-w. Good luck!
I remember that there is a distinct four-amino-acid insertion within the spike protein between S1 and S2, serving as a cleavage site for the protease furin. Published studies have discussed how this site is even proteolytically processed by various proteases (DOI: 10.1016/j.isci.2020.101212). If you included protease inhibitors in the lysis buffer, I doubt that cleavage occurs during cell lysis and instead suspect it happens during the cell culture phase. You may need to optimize your expression.
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