Extraction from Tissue

Note: The following procedure is used for extracting proteins from mouse brain and may be suitable for use with other soft tissues.

  1. Rapidly remove the tissue from the animal.
  2. Weigh tissue and record wet tissue weight. Cut the tissue into small pieces with a scalpel or a tissue slice blade.
  3. Transfer the tissue pieces into 5 volumes (w/v) of Buffer A (0.5 g tissue into 2.5 mL).
  4. Homogenize the tissue on ice using a homogenizer.
  5. Centrifuge the sample for 10 seconds at 9,500 x g in a microcentrifuge.
  6. Transfer the supernatant into a clean tube and determine the protein concentration by the Bradford method.
  7. Dilute the extract to 1 mg/mL in Buffer A.
  8. Use 1 mL of extract (1 mg/mL) for labeling with Cy3 or Cy5 dyes (see Sample Incubation on the Array).

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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