Bacterial Protein Extraction (mini-scale) Using B-Per
B-Per (Pierce, Cat number: 78248) Extraction of 1.5ml bact.culture OD600nm 1.5-3.5
A useful procedure for initial screening of expression conditions instead of sonication of bacteria; not recommended for large scale preparation of protein.
Not always the protein activity is conserved after B-Per extraction.
3. Spin 13000rpm 5min 4°C, to separate soluble proteins from the insoluble proteins in the pellet. Collect supernatant. Keep 40µl sample of supernatant for PAGE-SDS or western blot: soluble proteins.
4. Resuspend pellet in 300µl lysis buffer (25mM TrisHCl pH8.0; 0.1M NaCl; 10% glycerol; 0.1% Triton X-100 and 1mM PMSF). Spin 13000rpm 5min 4°C, to separate detergent-soluble proteins from inclusion bodies in the pellet. Collect supernatant. Keep 40ul sample of supernatant for PAGE-SDS or western blot: detergent-soluble proteins.
5. For inclusion bodies purification, add (lysozime 6µl of 10mg/ml) to the resuspended pellet to a f.c. of 200µg/ml, vortex 1min. Add 1ml of B-Per 1:10 to the suspension and vortex for another 1min. Collect inclusion bodies by centrifugation 13000rpm 10min 4°C. Resuspend pellet in 1ml of B-Per 1:10. Spin and repeat extraction again. Pellet can be resuspended in sample buffer for PAGE-SDS: inclusion bodies fraction or resuspended in denaturant buffer of Urea or Guanidine-HCl (see next point).
6. For inclusion bodies resuspension prepare lysis buffer (without detergent) containing 8M urea or 6M Guanidine-HCl. Resuspend cells in 300µl buffer + Urea or Guanidine-HCl by pipetting up and down until the cell suspension is homogenous. Spin 13000rpm 5min 4°C. Collect supernatant. Keep 40µl sample of supernatant for PAGE-SDS or western blot: inclusion bodies proteins. Precipitate Guanidine-HCl from samples with 9 volumes of ethanol (see protein precipitation protocol): resuspended Inclusion Bodies.
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