Freeze-thaw protocol
pH solubility screens
Salt solubility screen
Urea solubility screen
Detergents solubility screen
Stabilizer
Preparation of soluble/insoluble protein from cells
Jeanne Perry (Molecular Biology Institute, UCLA, Los Angeles, USA)
The solubility of a protein depends strongly on the composition of the lysis buffer. Using the procedure described below the solubility of a specific protein can be tested under many different conditions.
| 1. |
Grow the cells and induce protein expression using protocols described elsewhere. |
| 2. |
Take so many 1 ml-samples from the cell culture at OD600 of 1 as experiments you would like to carry out. If the OD of the cell culture is not 1 you have to correct the sample volume to obtain an equivalent size pellet. |
| 3. |
Spin down the cells for 5 min at 6000 rpm in a microfuge. |
| 4. |
To each cell pellet, add 100 ml of the appropriate buffer (see below) |
For a first screen use the buffer described in the table below.
| 7.5N |
50 mM Tris pH 7.5, 50 mM NaCl, 5 mM EDTA, 1 mg/ml lysozyme |
| 2S |
50 mM Tris pH 7.5, 2 M NaCl, 5 mM EDTA, 1 mg/ml lysozyme |
| 0.5U |
50 mM Tris pH 7.5, 50 mM NaCl, 5 mM EDTA, 0.5 M urea, 1 mg/ml lysozyme |
| D |
20 mM Tris pH 7.5, 50 mM NaCl, 0.2% NP 40, 1 mg/ml lysozyme |
| 5. |
Vortex to resuspend the cells. |
| 6. |
Sonicate (using a microtip) or Freeze-thaw (see below for protocol) to lyse cells. |
Freeze-thaw protocol
- freeze quickly on dry ice and leave for 3 min.
- thaw immediately at 42°C. Vortex vigorously to mix well.
- Repeat the two previous steps for three more times (4 freeze-thaw-vortex cycles in all).
| 7. |
Spin the tubes for 5 min at maximum speed in a microfuge. |
| 8. |
Separate the supernatant (contains soluble protein) from the pellet (contains insoluble protein) by pipetting out the supernatant to a clean tube. |
| 9. |
Label the supernatant "S__" (fill in with the buffer name, e.g. "S7N"). |
| 10. |
Label the pellet "P__" (fill in with the buffer name, e.g. "P7N"). |
| 11. |
To each supernatant, add 1 ml acetone and vortex. Freeze or leave on ice for 15 min. Spin 5 min at maimum speed. Remove the acetone by pipetting it out, being careful not to disturb the pellet. |
| 12. |
Dry at 37 °C. |
| 13. |
To the acetone-treated pellet, add 15 ml SDS-Page loading buffer. |
| 14. |
To the cell pellet, add 25 ml SDS-Page loading buffer. |
| 15. |
Heat all samples to 95 °C (or greater) for 5 minutes. |
| 16. |
Vortex and centrifuge 5 min at maximum speed. |
| 17. |
Load 10 ml on a SDS-PAGE gel, taking sample from the top and avoiding any pellet. |
Based on results, follow up by trying another set of screens and then continue with combinations.
pH solubility screens
| 5N |
50 mM Na Acetate pH 5, 50 mM NaCl, 5 mM EDTA, 1 mg/ml lysozyme |
| 6N |
50 mM MES pH 6, 50 mM NaCl, 5 mM EDTA, 1 mg/ml lysozyme |
| 7N |
50 mM Tris pH 7, 50 mM NaCl, 5 mM EDTA, 1 mg/ml lysozyme |
| 8N |
50 mM Tris pH 8, 50 mM NaCl, 5 mM EDTA, 1 mg/ml lysozyme |
| 9N |
50 mM Tris pH 9, 50 mM NaCl, 5 mM EDTA, 1 mg/ml lysozyme |
Salt solubility screen
| 0.1S |
50 mM Tris pH 7.5, 0.1 M NaCl, 5 mM EDTA, 1mg/ml lysozyme |
| 0.5S |
50 mM Tris pH 7.5, 0.5 M NaCl, 5 mM EDTA, 1mg/ml lysozyme |
| 1S |
50 mM Tris pH 7.5, 1 M NaCl, 5 mM EDTA, 1mg/ml lysozyme |
| 0.1KS |
50 mM Tris pH 7.5, 0.1 M KCl, 5 mM EDTA, 1mg/ml lysozyme |
| 1KS |
50 mM Tris pH 7.5, 1 M KCl, 5 mM EDTA, 1mg/ml lysozyme |
Urea solubility screen
| 0.5U |
50 mM Tris pH 7.5, 0.5 M Urea, 50 mM NaCl, 5 mM EDTA, 1 mg/ml lysozyme |
| 1U |
50 mM Tris pH 7.5, 1 M Urea, 50 mM NaCl, 5 mM EDTA, 1 mg/ml lysozyme |
| 2U |
50 mM Tris pH 7.5, 2 M Urea, 50 mM NaCl, 5 mM EDTA, 1 mg/ml lysozyme |
| 3U |
50 mM Tris pH 7.5, 3 M Urea, 50 mM NaCl, 5 mM EDTA, 1 mg/ml lysozyme |
| 4U |
50 mM Tris pH 7.5, 4 M Urea, 50 mM NaCl, 5 mM EDTA |
| 5U |
50 mM Tris pH 7.5, 5 M Urea, 50 mM NaCl, 5 mM EDTA |
| 6U |
50 mM Tris pH 7.5, 6 M Urea, 50 mM NaCl, 5 mM EDTA |
Detergents solubility screen
| D |
20mM Tris HCl pH 7.5, 50 mM NaCl, 0.2% NP 40, 1 mg/ml lysozyme |
| X |
20mM Tris HCl pH 7.5, 50 mM NaCl, 0.2% triton X-100, 1 mg/ml lysozyme |
| T |
20mM Tris HCl pH 7.5, 50 mM NaCl, 0.2% Tween-20, 1 mg/ml lysozyme |
| M |
20mM Tris HCl pH 7.5, 50 mM NaCl, 0.2% dodecylmaltoside, 1 mg/ml lysozyme |
Stabilizer
| 10G |
20mM Tris HCl pH7.5, 50 mM NaCl, 10% glycerol, 1 mg/ml lysozyme |
| 50G |
20mM Tris HCl pH7.5, 50 mM NaCl, 50% glycerol, 1 mg/ml lysozyme |
