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.
1. Spin bacterial cells 10min 5000rpm in a microcentrifuge 4°C (cells can either be used fresh or frozen at -70°C).
2. Resuspend cells in 300µl of B-Per reagent (Pierce) by either vigorous vortexing or by pipetting up and down until the cell suspension is homogenous. Vortex for 1 more min. (if suspension is too viscous add Dnase 100U/ml or 25-50µg/ml (SIGMA DN-25). Incubate 10min 4°C in the presence of 10mMMgCl2
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.
7. You can try first 8M of Urea, and if protein is soluble titer down in the next experiments the urea concentration till minimal urea is required for protein solubilization.
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