What kinds of detergent-free bacterial lysis buffers exist? The proteins we're extracting will be later analyzed by LC-MS/MS, and we're looking for a lysis buffer that won't interfere with this downstream analysis.
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4 Answers
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I'm a HUGE fan of FASP (filter-aided sample preparation) which is an in-solution preparation/digestion. It is very fast, allows you to not worry about a protein precipitation step, and gets rid of all MS incompatible substances with the wash steps (so doesn't matter what lysis buffer you use). It requires very few, inexpensive materials (a pack of 100 centrifuge filter units is less than 250 USD from VWR) and has great recovery. This is the link to the Nature Methods paper that the linked protocol is based on (the linked protocol is an updated, faster version of the Nature Methods one).
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There are a few techniques available for this.
Some recommend a urea-based technique, others recommend a chloroform. Do you have more information about your specific needs?
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why not use a french press? put a few thousand PSI on the sample and the cells just pop open. The lysis buffer contains no detergents:
Lysis buffer:
50 mM Tris-HCl pH 7.5
50-200 mM NaCl*
1 mM MgCl2
5 mM DTT
1 mM PMSF
the salt concentration is variable and you could probably do without the DTT too if thats a problem ...
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As pstew mentioned, SDS based lysis followed by FASP is a very good way. However, FASP can lead to sample losses. I would use it only if you have large culture of bacterial cells.
If you want totally detergent free method then, lyse cells in 8 M guanidinium hydrochloride buffered to pH 7.5-8.2 using 50 mM Tris buffer or 50 mM freshly prepared ammonium bicarbonate. If your cell pellet is approximately 20 uL then add 60 uL 8 M GuHCL buffered solution so that final conc of GuHCL is ~ 6M, heat to 65 degrees C and shake for 30 minutes or so. Spin down to remove any insoluble debris. Measure protein concentraion, if you want to, using BCA assay. Then, reduce with 10 mM DTT for 30 minutes at 56 C, alkylate with 25 mM iodoacetamide at room temperature and in dark (for this step it is crucial that the pH of your sample is around 8.0 otherwise iodoacetamide will cause side reactions). Dilute at least 8 times and digest overnight with Trypsin.
