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Upstate - Procedures and Protocols - Preparation of Brain Cytosol
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Upstate Cell Signaling Symposium
The first annual Upstate Cell Signalling Symposium will take place in Dundee, Scotland (UK) from 6 - 9th June 2004 and is entitled:

Regulation and Therapeutic Potential of the PI 3-kinase/PKB Signalling Pathway.

The symposium is co-sponsored by Sir Philip Cohen and Prof. Dario Alessi (The MRC Protein Phosphorylation Unit) in conjunction with Upstate.

Participation is strictly limited to 300 delegates, so early registration is recommended.

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.

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Preparation of Modifed Radioimmunoprecipitation (RIPA) Buffer

Activation of Sodium Orthovanadate

Preparation of Cell Lysate

Western Immunoblotting of Proteins

Troubleshooting Tips for Western Immunoblotting

Immunoprecipitation of Proteins

Immunoprecipitation / Kinase Assay

Troubleshooting Tips for Immunoprecipitation / Kinase Assays

Assay for Immunoprecipitated Phosphoinositide 3-Kinase (PI 3 Kinase) Activity (in vitro)

Phospholipid Vesicle Preparation

Chromatin Immunoprecipitation (ChIPs) Assay Kit

Forskolin Treatment of Cells

Preparation of Brain Cytosol

Immunohistochemistry with Fixed Paraffin-Embedded Tissue Sections

Tips for Immunohistochemistry with Fixed Paraffin-Embedded Tissues

Immunohistochemical Methods to Detect Nitrotyrosine

Immunocytochemistry with Adherent Cells

Differentiation of Stages in the Cell Cycle

Elution of GST-Fusion Protein from Glutathione Agarose

Purification of Antiserum or Ascites by Protein A/G Chromatography

Molecular Weights of Some Common Proteins

Transient Transfection of Cos-1 Cells

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