Magnesium acetate in protein lysates - (Mar/21/2012 )
I'm working on preparation of protein lysates from recombinant E.coli. I induced 1OD culture with 0.5mM IPTG and grew it for 2 hours. After harvesting the cells, I used a lysing solution (Tris, NaCl, EDTA, lysozyme) and then sonicated the sample. When I ran the cell lysate on 10%SDS PAGE, I can't see a large blob-like band for my over-expressed protein! Infact, I can't even locate my over-expressed protein properly!!!
I read in a few articles that magnesium acetate should be added before sonication. What is the role of magnesium acetate???!!! I'm so confused!!!
Please help me out
I don't know the function of magnesium acetate for sonication. I did a quick Google search to find articles on the subject and came up with nothing.
I have successfully used sonication to lyse E. coli after overexpressing proteins. Make sure the sonicator has been tuned properly before every run. I usually do 10 repetitions of the following:
10 second sonicator burst at the highest setting for your particular sonifier probe with 30 seconds on ice to cool the sample. Repeat 9 times. This should lyse most of the cells especially after your lysosome step.
If the sonification doesn't work, try chemical lysis. Novagen (EMD Biochemicals) makes a great product called Bugbuster to lyse E.coli. It is REALLY easy to use with nice results. If you go this route, make sure to purchase the kit with Benzonase. This enzyme will chew up DNA and RNA, leaving only the proteins behind.