Hello, currently I am doing a recombinant protein expression on E. coli BL21. For my research I am checking the extracellular for the targeted protein. Currently the expression and purification so far yield a good result with a band around the expected kDa. However when I am doing a dialysis for the next step (activity assay) for some reason the protein got partially degraded I think? The kDa for some reason got lower, after checking the SDS PAGE result these only happen after dialysis for the extracellular, my research partner who is checking the periplasm section for the targeted protein didn't have any problem with the protein and still have the expected kDa after dialysis. Do anyone know why this happen? I had tried searching for troubleshoot but so far nothing seems to make sense.
Protease activity? How pure is your preparation?
To test this hypothesis, you could check at different time points.
Dear Martinus
The size reduction could be due to the removal of extracellular signal peptide by a protease. For that purpose, check how much reduction in protein size occurs. Is the reduction in size constant every time after dialysis or random?
Secondly, if the protein is getting degraded, try adding PMSF and EDTA in you protein solution to prevent the degradation.
Try adding a protease inhibitor cocktail to the sample prior to dialysis.
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