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Microsomal Preparations for Western Blotting

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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