Hi Everyone! I have a sample of bacteria cells, and I want to sonicate them to extract cytosolic proteins. I found a protocol using Tris-HCl buffer, but this lab did not use proteases inhibitor. Does anyone have a protocol or know a good website to look into? I just want to measure the total protein in the cell.
Thanks in advance!
Hi! I can share with you the lysis protocol I regularly use for protein purification from E.coli. Since you're not purifying and just want to see cytosolic proteins, I guess it'll work.
Lysis Buffer
Sodium phosphate 50 mM
NaCl 300 mM
Lisozyme 1 mg/mL
SIGMAFAST™ Protease Inhibitor Cocktail Tablets, EDTA-Free, half a tablet
pH 7.4
I recommend to use 4-5 mL lysis buffer per g of bacteria pellet.
If you don't have the inhibitor tablets you could use 1 mM PMSF (phenylmethylsulfonyl fluoride).
Cheers and good luck!
Hello
I will recommend that you have a look at the " Methods in Enzymology" Volume 22 It has very useful information & protocol related to you question.
Good Luck.
I often use Tris-HCl buffer + 1% Triton X-100 + 1% PMSF (as a protease inhibitor), and use BCA for protein quantification after sonication
- Badges
- Science topic