Extraction from Cells

From adherent cells:

  1. Grow cells to 70-80% confluence. (2-3 Petri dishes of 10 cm will give enough material for labeling.)
  2. Wash the cells twice with cold 0.01 M PBS pH 7.4.
  3. Add 1 mL of Buffer A directly onto each plate. Incubate for 5 minutes on ice. Scrape the plate with a rubber policeman and collect the sample into a microcentrifuge tube.
  4. Proceed to step 4 for Non-adherent cells.

From non-adherent cells:

  1. Grow cells in culture. Collect 107 cells into a test tube. Centrifuge the cells at 300 x g for 5 minutes.
  2. Wash the cells twice with cold 0.01M PBS, pH 7.4, and collect by centrifugation at 300 x g.
  3. Transfer the cells to a microcentrifuge tube, add 1 mL of Buffer A, and vortex. Incubate for 5 minutes on ice.
  4. Centrifuge the sample for 10 seconds at 9,500 x g (in a microcentrifuge). Transfer the supernatant to a new microcentrifuge tube.
  5. Determine the protein concentration in the supernatant by the Bradford method.
  6. Dilute the sample to 1 mg/mL of protein in Buffer A.
  7. Use 1 mL of extract (1 mg/mL) for labeling with Cy3 or Cy5 (see Sample Labeling and Processing).

Dialysis

Before performing the labeling procedure (see Sample Labeling and Processing) it is important to bring the extract to the required pH by dialysis.

  1. Prepare 0.1M, pH 9.5-9.6, Carbonate-Bicarbonate Buffer (dissolve 2 capsules of Product No. C3041 into 100 mL water).
  2. Dialyze at 4 °C for 2 hours in a dialysis buffer volume 1000 times the volume of the nuclear protein extract.
  3. Replace the dialysis buffer to a freshly prepared carbonate buffer and dialyze for an additional 2 hours, at 4 °C. Determined protein concentration according to the Bradford procedure.

Materials

     
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