An alternative experimental approach to the isolation of crude plasma membrane fractions from tissues
An alternative experimental approach to the isolation of crude plasma membrane fractions from tissues
The method described by Neville (1968) works well for isolating murine liver plasma membranes, but is not necessarily applicable for isolating plasma membranes from other tissues. Here, a method for preparing crude plasma membrane fractions from other tissues is described. This experiment was derived from the Experimental Guide to Protein Purification and Identification by Houzhu Zhu.
Operation method
An alternative experimental approach to the isolation of crude plasma membrane fractions from tissues
Materials and Instruments
Homogenizing Buffer Move Equipment For more product details, please visit Aladdin Scientific website.
Dounce's Glass Homogenizer (15-ml) with A pestle and mortar
Dounce's glass homogenizer (15-ml) with A pestle and mortar
Reagents
Homogenization buffer
(for recipe, see Preparation of Reagents PP.234-240)
Procedure
All operations are carried out at 0 to 4°C.
1) Chop tissue pieces on ice-cold homogenization buffer plants and pour off bleed water. Rinse the tissue pieces with homogenization buffer and place them on ice. Repeat until the tissue is broken into 1 mm3 pieces and there is no visible blood.
2) Homogenize the tissue by adding 5 times (v/v) the volume of homogenizing buffer to the volume of the tissue pieces and pumping 10-20 times with a pestle and mortar in a Dounce's glass homogenizer in an ice bath.
3) Homogenize by centrifugation at 4°C for 10 min at 600 g. The supernatant contains the cell membranes, mitochondria, and cytosol, while the precipitate contains the unbroken cells and the nuclei. The supernatant contains cell membrane, mitochondria and cytosol, and the precipitate contains unbroken cells and nuclei. Discard the precipitate.
4) Centrifuge the supernatant at 8000 g for 10 min and precipitate the mitochondria. Discard the precipitate.
5) Centrifuge the supernatant at 100,000 g for 20 min at 4°C using a rotary head.
6) Resuspend the precipitate in homogenization buffer. Homogenize in a small Dounce's homogenizer and centrifuge again at 100,000 g for 20 min at 4°C. Discard the supernatant.
7) Resuspend the precipitate thoroughly with a small volume of appropriate buffer.
8) Take a small aliquot of the membrane for protein measurement. The crude membrane can be snap frozen in a dry ice/ethanol bath and stored at -85°C.