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Protein PurificationExtraction and Clarification

Solubility Studies

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