Preparation of Brain Cytosol
1. Chill a glass plate on ice.
2. Place brain tissue on the chilled glass plate and cut slices of brain into small pieces.
3. Resuspend the pieces in ice-cold RIPA buffer without the detergents NP-40 and Na deoxycholate. Use 1L per 250 g of tissue
4. Perform the following step in a cold room: Transfer the suspended tissue to a blender and homogenize on low power with four 30 second bursts. Let the homogenate cool for one minute between bursts. Repeat bursts until the homogenate is smooth.
5. Transfer the homogenate to pre-chilled centrifuge tubes.
6. Centrifuge at 2900 x g for 20 minutes at 4íC. This step sediments unbroken cells and nuclei. Discard the pellet.
7. Transfer the supernatant fraction to another centrifuge tube.
8. Centrifuge this supernatant fraction at 29,000 x g for 45 minutes at 4íC.
9. Transfer and save the supernatant fraction. This fraction contains brain cytosol and microsomal membranes. Concentrate the brain cytosol proteins by precipitation with glacial acetic acid as described below.
10. Resuspend the pellet in 10 ml of ice-cold RIPA buffer containing detergents (please see protocol "Preparation of Modified RIPA Buffer").
11. Centrifuge the resuspended pellet at 15,000 x g for 20 minutes at 4íC.
12. Transfer the supernatant fraction to another tube. This fraction contains solubilized mitochondrial and plasma membrane proteins.
Concentration of Brain Cytosolic Proteins by Precipitation with Glacial Acetic Acid
CAUTION: Glacial acetic acid is caustic. Perform the following step in a hood and wear gloves, goggles, and a lab coat.
1. Acidify the brain cytosol fraction (Step 9 above) to pH 4.5 by slowly adding and mixing drops of glacial acetic acid.
2. Sediment the precipitated proteins by centrifuging the acidified sample at 15,000 x g for 20 minutes at 4íC. Discard the supernatant fraction.
3. Dissolve the pellet in 10 ml of desired buffer.
4. Determine protein concentration by a standard method.