The membrane homogenate preparation procedure isolates cell membranes and membrane-associated macromolecules and assures their homogeneity for assaying small fractions of the preparation in each of many test tubes. It removes the majority of soluble macromolecules, small molecules (including endogenous receptor ligands), and minerals via multiple centrifugation steps each followed by resuspension in fresh buffer containing the divalent ion chelator ethylenediamine tetraacetic acid (EDTA). Some prefer a P2 preparation, which involves discarding an initial pellet generated by a low-speed centrifugation step, but this appears to be unnecessary for these assays.
In the case that the membranes will be assayed for agonist-stimulated [35S]GTPyS binding, a preincubation step to remove endogenous adenosine is useful. It has been noted that a significant quantity of endogenous adenosine remains in membrane preparations of brain tissue, which causes elevation of basal [35S]GTPyS binding, thereby reducing the signal-to-noise ratio for agonist-stimulated [35S]GTPyS binding for other agonists. To eliminate this complication, membranes can be routinely pretreated with adenosine deaminase to remove this adenosine and its accompanying activity. A stock of adenosine deaminase of 1-2 U/mL can be kept in 1 mg/mL BSA at -4°C for months at a time.
For either agonist-stimulated [35S]GTPyS binding or receptor binding when endocannabinoids, like 2-arachidonyl glycerol (2-AG) or anandamide, are to be assayed, pretreatment of the membranes with phenylmethylsulfonyl fluoride (PMSF) is advisable. PMSF irreversibly inhibits amidase enzymes, including fatty acid amide hydrolase (FAAH), the enzyme that degrades endocannabi-noids. This treatment has been shown to greatly increase the potency of endo-cannabinoids in such assays. A stock solution of PMSF is prepared immediately before use at 100 mM in «-butanol.
Specific CNS regions of interest or whole brain and/or spinal cord are dissected from freshly sacrificed animals on ice. The preparation can be handled in one of two ways: tissue can be frozen at -80°C at this point until the day it is to be assayed, or membrane homogenates may be prepared prior to freezing and the homogenate separated into aliquots and frozen until the day they are to be assayed (see Note 1). To prepare membrane homogenates from cultured cells, cell media is replaced with cold membrane buffer (50 mM Tris, pH 7.4, 3.0 mM MgCl2, 0.2 mM EGTA), and the cells are scraped or triturated from their growing surface and transferred to centrifuge tubes. In any case (whether preparing tissue or cultured cells and whether the tissue is prepared immediately after dissection or after frozen storage), each sample is placed in a centrifuge tube with 3-15 mL of cold membrane buffer. Samples are kept on ice or in the refrigerated centrifuge throughout the membrane preparation procedure.
1. Pulverize each sample with an electric tissue grinder for 30 s until homogeneous. As an alternative to the electric grinder, a ground glass homogenizer can be used and is especially useful for very small samples, since a small but significant amount of tissue is lost each time the electric grinder is used.
2. Centrifuge samples at 40,000g for 10 min at 4°C.
3. Discard the supernatant, then resuspend the pellet with the tissue grinder for 10 s in 3-15 mL of cold membrane buffer.
4. Centrifuge and resuspend the pellet again as before.
5. Repeat Steps 2 and 3. If endocannabinoids are being assayed, the membrane homogenate should be treated by adding PMSF to a final concentration of 50 |M and thoroughly mixing (see above). No additional incubation should be necessary, so the homogenate can undergo the final centrifugation (same conditions as before) almost immediately.
6. The final resuspension should be in a volume calculated to give an appropriate protein concentration; approx 100 mg of membrane protein is obtained for every gram of brain tissue prepared. After the final resuspension, a sample of the homogenate is taken for determination of protein content by Bradford or Lowry assays.
7. If the sample was not frozen prior to membrane preparation, it may now be separated into aliquots for storage at -80°C. On the day of the binding assay, thawed aliquots are resuspended with the tissue grinder for 10 s in the desired amount of buffer and the protein content determined.
8. If the membranes are to be assayed for [35S]GTPyS binding, add adenosine deaminase from a stock solution to give a final concentration of 0.004 U/mL, and incubate the membranes for 10 min at 30°C.
9. The membrane homogenate is now ready to be added to a binding assay.
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