[Protein-analysis] Ecol expressed Calmodulin doesn't work?

Isaac Li isaac.li at utoronto.ca
Wed Apr 12 12:46:27 EST 2006


Hi, My name is Isaac, I just entered the field of biology. I have been 
trying to express human calmodulin in E.coli cells, and tried a binding 
assay for its activities but it doesn't work, can anyone give me some 
advices?

My CaM extraction procedures:
1. express Calmodulin-CFP-GST fusion in DH5a, 37 degrees, HB 
broth+ampicillin for 24 hours.
2. I sonicated the cells in Tris buffer
3. I span down the cell debris, and take the lysate to check for CaM 
activity.

What is the commonly used assay to test CaM binding to peptide? and how 
do we usually get CaM working? I sorta recall that CaM folds really 
well... so i don't think it would be a folding problem..
Thank you very much

Isaac


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