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290 Tchaga
3. Centrifuge the tube again (for ∼2 min) and aspirate any residual traces of liquid.
Reweigh the tube to determine the weight of the cell pellet.
4. Freeze your samples by placing them in liquid nitrogen or in a –80ºC freezer.
5. Re-suspend the cell pellet (∼100 mg) in 30 μl of Buffer A for each mg of cells
(see Note 3).
6. Disperse the pellet by gently pipetting up and down approximately 20 times.
7. Incubate at 4ºC for 10 min, inverting the tube every minute during incubation.
Transfer the cell lysate to a microcentrifuge tube.
8. Centrifuge the cell extract at 10,000 × g for 20 min at 4ºC to remove insoluble
material (see Note 4).
9. Transfer the supernatant to a clean tube without disturbing the pellet. This is the
starting clarified sample used in the PMAC chromatography.
10. Reserve a small portion of the clarified sample at 4ºC for phosphate, protein, and
other analysis (see Note 5). Proceed to Subheading 3.3.
3.2. Extracting Protein from Crude Tissue
1. Before starting, chill the following items on ice or at 4ºC.
• 5 ml Buffer A.
• one mortar & pestle.
• two 2-ml microcentrifuge tubes.
• one 5-ml tube.
2. Transfer 100–200 mg of frozen tissue to a pre-chilled mortar.
3. Add 0.25–0.5 g of Alumina to the mortar.
4. Use the pestle to grind the tissue until a paste is formed.
5. Add 2 ml of pre-chilled Buffer A.
6. Mix the buffer into the paste using the pestle. When complete, use a micropipette
tip or sterile instrument to scrape any paste that adheres to the pestle back into
the mortar.
7. Transfer the extract to a pre-chilled 2-ml microcentrifuge tube.
8. While holding the pestle over the mortar, rinse the pestle with 2 ml of Buffer A
pre-chilled at 4ºC.
9. Combine the rinse with the original extract in a 2-ml tube. (Use a second 2-ml
tube if the volume exceeds the tube’s capacity.)
10. Centrifuge the suspension at 10,000 × g and 4ºC for 20 min (see Note 6).
11. While taking care not to disturb the pellet, transfer the supernatant to a pre-chilled
5-ml tube.
12. Gently invert the tube to mix the lysate (see Note 7).
13. Reserve a small portion of the clarified sample at 4ºC for phosphate,
protein, and other analysis. Proceed to Subheading 3.3.: Column Enrichment
(see Note 8).
3.3. Column Enrichment
1. Allow the column to stand at room temperature in an upright position until the
resin settles out of suspension.