Bacterial Protein Extraction - mini scale - Sonication

互联网2013-09-06

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<center> Bacterial Protein Extraction - mini scale - Sonication </center>

Protein Extraction

1) Resuspend pellet of 10ml cell culture in 1ml lysis buffer (or 100ml bacterial culture for very low expression level).

Suggested Lysis buffer : 140mM NaCl; 2.7mM KCL; 10mM Na₂ HPO₄ ; 1.8mM KH₂ PO_4; pH 7.3 (PBS)
                                    or 100mM NaCl; 25mM TrisHCl; pH 8.0
                                    optional 0.02% NaN₃ (azide)
                                    optional protease inhibitors

Optional additives to the lysis buffer
a) 1mM PMSF or protease inhibitor cocktail 1:200 (cocktail for bacterial cells #P-8849 from Sigma)
b) Dnase 100U/ml or 25-50ug/ml (SIGMA DN-25). Incubate 10min 4°C in the presence of 10mMMgCl₂ .
c) Lysozime 0.2mg/ml. Incubate 10min 4°C.
d) ßME, DTT or DTE up to 10mM for proteins with many cysteines.
e) 0.1-2% Triton X-100, NP40; or any other detergent that do not affect the biological activity of your protein.
f) 10% glycerol (for stabilization of the protein and prevention of aggregation).

2) Sonicate in ice bucket 3 x 10sec or more if the cells are not completely disrupted (Lysis is complete when the cloudy cell suspension becomes translucent. Avoid protein denaturation by frothing).

3) Spin 5min 13000rpm 4°C. Separate soluble proteins (supernatant) from insoluble or inclusion bodies proteins (pellet). Use supernatant for next step. Keep sample of 40ul of supernatant for PAGE-SDS and Western blot: soluble proteins

4) Resuspend pellet in another 1ml lysis buffer and keep sample of 40ul for PAGE-SDS and Western blot: insoluble proteins, or unlysed cells .

Analysis of results - Troubleshooting

Expect over-expressed protein to be found only in the crude supernatant.
If most of the protein remains insoluble after extraction, try
a) To change lysis buffer by adding ßME, DTT, glycerol, detergents or more NaCl. If only part of it is insoluble,
b) Or re-extract pellet with more buffer,
c) Or use more lysis buffer during extraction,
d) Or perform a more intensive sonication,
e) Or incubate with lysozyme before sonication.
f) Or try the denaturating protocol extraction (4-6 M guanidine-HCl; 4-8 M urea; alkaline pH>9.0; 0.5-2% Triton X-100; 0.5-2% N-lauroylsarcosine or other detergents)
g) See Solubility Studies: Preparation of soluble/insoluble protein from cells according to the PROTEIN EXPRESSION AND PURIFICATION FACILITY of the EUROPEAN MOLECULAR BIOLOGY LABORATORY