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P r o t o c o l s

Preparation of Brain Membrane Fractions for Western Blot Analysis

All the procedures should be done at 4C using precooled reagents. For rat samples, immediately remove brain from the cranium into ice cold dissection buffer (50 mM Tris-acetate (TA), pH 7.4, 10% sucrose, 5 mM EDTA) containing a freshly added protease inhibitor cocktail of 1 mM phenylmethylsulfonyl fluoride, 20 g/ml benzamidine, and 20 g/ml iodoacetamide. For monkey or human brain, remove tissue no later than 46 hr postmortem and process as described for the rat tissue. Different parts of the brain can be dissected out and the enriched plasma membrane can be prepared as described below.

I. Synaptic Plasma Membrane

  1. Long Protocol: This protocol is adapted from Rogers et al., 1991, J.Neuroscience: 2713-2724. It should be followed when an enriched membrane preparation and postsynaptic densities are desired.
    1. Add 9 volumes of dissection buffer to the tissues and homogenize in a motor driven glass-teflon homogenizer at 900 rpm (1215 strokes). Never use a polytron.
    2. Centrifuge the homogenate at 800 x g for 20 min.
    3. Spin resulting supernatant at 16,000 x g for 30 min.
    4. Resuspend the pellet in 20 mM HEPES buffer (pH 7.4), rehomogenize and centrifuge at 16,000 x g for 15 min.
    5. Pool the pellets from Step 2 and Step 4, resuspend in 5 vol (of the original tissue weight) of lysis buffer (5 mM Tris-acetate, pH 8.1, and freshly added protease inhibitors as above).
    6. Gently homogenize with three strokes of the piston or pipetting up and down in a glass pipette.
    7. Leave the homogenate on ice for 45 min to lyse synaptosomes and vesicles.
    8. Homogenize again with 10 strokes of the piston.
    9. Supplement the homogenate to get a final concentration of 34% (w/w) sucrose and 5 mM Tris-acetate, pH 7.4.
    10. Use this homogenate as a bottom layer of three-step gradient. Carefully overlay with equal volume of 28.5% (w/w) sucrose/TA and a one-third volume of 10% sucrose (w/w)/TA.
    11. Centrifuge the gradient at 60,000 x g for 2 hr.
    12. Collect the broad band at the 28.5% and 34% sucrose interface. This consists primarily of synaptic plasma membrane.
    13. Dilute with TA to 10% sucrose (by adding 3 vol of TA), and pellet by centrifugation at 48,000 x g for 30 min.
    14. Resuspend the pellet in water or TA, estimate the protein concentration, aliquot, and store at 70C.
    15. Use 2050 g protein per lane of mini SDS-polyacrylamide gels or 50100 g per lane of standard gel.
    16. Use any standard western blot protocol for immunodetection of glutamate receptor protein.
  2. Short Protocol: This is a time-saving protocol; however, it yields a crude membrane preparation.
    1. To each tissue add 9 volume of dissecting buffer and homogenize in a motor-driven glass homogenizer at 900 rpm (never use polytron) for 1215 strokes.
    2. Centrifuge the homogenate at 800 x g for 20 min.
    3. Remove the supernatant, homogenize again, and centrifuge at 16,000 x g for 30 min.
    4. Resuspend the resulting pellets in 10 ml 320 mM sucrose by pipetting up and down and centrifuge at 9200 x g for 15 min.
    5. Resuspend resulting pellets in a total volume of 15 ml 320 mM sucrose, add 9 volumes of water and immediately homogenize in glass-teflon homogenizer (3 strokes at 1500 rpm). Proceed by adding the previously mentioned protease inhibitors. Incubate on ice for 1530 min.
    6. Spin at 25,000 x g for 20 min.
    7. Resuspend pellets in approximately 5 ml water. Add sucrose and water to give a final volume of 45 ml 1.2 M sucrose, add HEPES to 5 mM and the protease inhibitors as above. Homogenize in glass teflon-homogenizer (3 strokes, 1000 rpm).
    8. Spin at 9200 x g for 20 min.
    9. Resuspend pellets in TA or water. Estimate the protein concentration. Run 2050 g protein per lane of mini SDS-polyacrylamide gels or 50100 g per lane of standard gel.
    10. Use any standard western blot protocol for immunodetection of glutamate receptor protein.

II. Post Synaptic Densities

  1. Resuspend the synaptic plasma membranes (from Step 14, Long protocol) in 3 ml TA, homogenize in a Dounce homogenizer.
  2. Mix with an equal volume of 3% Triton X-100, and leave on ice for 30 min. with occasional mixing.
  3. Layer on 30 ml of 28.5% (w/w) sucrose/TA. Centrifuge at 105,000 x g for 1 hr.
  4. Resuspend the pellet containing postsynaptic densities in water or TA, estimate the protein concentration, aliquot and store at -70C.
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Preparation and Staining of Frozen Tissue Sections
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Microwave citrate Pretreatment of Paraffin Sections
Immunohistochemistry / Tissue Section Staining
Cell Staining for Immunofluorescence Microscopy
Western Blotting
Western Blotting with Alkaline Phosphatase Conjugates
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Western Blotting with Monoclonal Antibodies
Western Blotting with Rabbit Polyclonal Antibodies
Preparation of Brain Membrane Fractions for Western Blot Analysis
Immunopurification of Tyrosine Phosphorylated Proteins
Immunoprecipitation with Antibody:Agarose Conjugates
Immunoprecipitation with anti-Phosphotyrosine: Biotin Conjugates
Immunoprecipitation With Soluble Antibodies
Immunoprecipitation of 35S Labeled Cells
RNAse Protection Assay (RPA)
RNase Protection Assay
Total RNA Isolation
Protein Kinase Assay (PKA)
Protein Kinase Assay with an Immunocomplex
Protein Kinase Assay after Denaturation and Renaturation

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