B-PER BACTERIAL PROTEIN EXTRACTION REAGENT |
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Last Update: December 2006 |
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| PROCEDURE | |
| NOTE: this product can be obtained from Pierce. For some applications, it can substitute other means of cell lysis for protein purification, such as the French Press or Sonication methods (see protocol 111. GST-protein fusion expression and purification) | |
| In summary, it is very simple and quick. Trying this reagent before using more complex and time consuming methods is advisable since it tends to work well with most proteins | |
| Pierce NOTE: This reagent utilizes a proprietary, mild, nonionic detergent in 20 mM Tris-HCl (pH 7.5) for lysis of the bacterial cells. This method eliminates the need for mechanical disruption and eliminates exogenous contamination of the recombinant protein by the lysis reagent. Depending on the particular application, additional components such as lysozyme, protease inhibitors, salts, reducing agents, chelating agents, etc. may be added to the reagent. The reagent may be used for both soluble and insoluble protein extraction and inclusion body purification from total bacterial cell lysates | |
1. Pellet induced bacterial cells by centrifugation at 5,000 rpms for 10 minutes |
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| 2. Freeze cells at -70oC (this helps in lysis). Cells can be stored for day | |
| 3. Resuspend cells in ~5 mL of B-PER reagent for every 50 mL of culture (vortex or pippet up and down until a homogeneous mixture is achieved). | |
| NOTE: start with smaller volumes of B-PER and try to resuspend the pellet. Add more reagent if the pellet does not resuspend. Do not use too much reagent or the protein solution will be too dilute | |
| 4. Shake solution for an additional ~10 minutes at room temperature | |
5. Centrifuge at 27,000 xg for 15 minutes |
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| OPTIONAL: Pierce has a protocol for inclusion body purification using this reagent. | |
| 6. Take the soluble fraction and use for further purification | |