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| Here you find two protocols for the preparation of microsomes for western blotting: 1. from tissues, 2. from cell lines. From Tissues:Perform all operations at 4 °C 1. Homogenize tissue in 3 volumes of 20 mM potassium phosphate buffer pH 7.4, containing 0.1 mM EDTA, and 135 mM KCl. 2. Centrifuge homogenate at 10,000 g for 20 min. 3. Centrifuge the supernatant at 150,000 g for 1 hour. 4. Resuspend the micosomal pellet in 20 mM potassium phosphate buffer pH 7.4, containing 1 mM KCl and 10 mM ETDA. 5. Assay the preparation using a Bradford assay, and adjust the protein concentration to 1.0 mg/mL. 6. Run a total of 25 µg of total protein on SDS-PAGE using the procedure described by Laemmli, U.K. (1970) Nature 227: 680-685. From Cell Cultures:1. Wash cells 3 times with 2 mL cold solution containing 8 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl and subsequently suspended in 1.5 mL of this buffer. 2. Centrifuge cells at 360 g for 2 minutes at 4 °C and suspend in 1 mL of 100 mM potassium phosphate buffer pH 7.4, containing 10 mM ETDA. 3. Sonicate the cell suspension for 20 x 1 sec. 4. Centrifuge the cell lysate at 10,700 g for 60 minutes at 4 °C. 5. Homogenize the pellet in 100-150 µL 50 mM potassium phosphate buffer pH 7.4, containing 0.1 mM EDTA, and 10% (v/v) glycerol. 6. Determine protein concentration using a Bradford assay (usually 100-400 µg protein/dish is recovered). These protocols can be downloaded (pdf, 93 kB). |
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