Hi please suggest me a procedure of sample preparation for native PAGE. I am working on Gram Positive Bacteria that is highly resistant. I tried to break this with sonication using very high frequency but was not able to broke. I also try using freeze and thaw with liquid nitrogen and hot water but again failed. Triton X-100 and other non ionic detergents are also ineffective. These cells can be break by either SDS or 8 M Urea, but the problem is that i have to do Native PAGE. Please suggest me how i can break these cells. Protein of interest is of 8 Kd.
You could try using a French Press or a bead beater, combined with lysozyme pre-treatment.
Sir I tried with bead beater but was not successful to break and i dont have french press.
if you want to break gram positive bacteria, you should use lysozyme treatment before french press or sonication. if lysozyme turns out ineffective try lysostaphin.
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