Bacterial Protein Extraction (mini-scale) Using B-Per

互联网2013-09-06

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<center> 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 </center>

<center> 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. </center>

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 25-50 µg/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.