what is brain membrean structure
Answers
Brain Membranes.
Brain membranes are incubated on ice in 15-ml Pyrex Sorvall tubes (2-mm-thick Pyrex, 18 × 100 mm; DuPont, Wilmington, DE) in the presence of buffer alone (10 mM Tris, pH 7.4) or buffer containing drug in a total volume of 100 μl for 30 min. 125I-6-AIPP-Fsk or 125I-7-AIPP-Fsk is added to the membranes to a final concentration of approximately 3 nM. The labels are added directly from the ethanol solutions, and the final concentration of ethanol in the assays does not exceed 2% (v/v). The membranes are incubated for an additional 30 min on ice in the dark. Immediately before photolysis, 0.9 ml of the corresponding buffer solution containing the drug used for competition is added, and the diluted samples are quickly photolyzed (5 sec) in an ice–water bath at a distance of 10 cm from a high-pressure 1-kW AH-6 mercury lamp. Alternatively, samples can be photolyzed in microcentrifuge tubes in a Stratagene (La Jolla, CA) UV Stratalinker (energy, 200,000 μJ) for 1 min. Immediately after photolysis, 10 μl of 2-mercaptoethanol (final concentration of 1%, v/v) is added to each tube as a scavenger for long-lived reactive species. The membranes are centrifuged at 16,000g for 15 min at 4°, and the supernatants are removed. Membrane pellets are resuspended in SDS–PAGE sample buffer. After SDS–PAGE on an 10% (w/v) gel, the labeled proteins are detected by autoradiography.
As can be seen in Fig. 3, a number of protein bands are labeled by 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk in the absence of any competitors and can be observed after overnight exposure of the dried gels to X-ray film (Fig. 3, lanes marked None). The specificity of labeling is determined by preincubating the membranes with buffer, 100 μM forskolin, 100 μM 1,9-dideoxyforskolin, or 100 μM cytochalasin B and then with the photoaffinity labels. 125I-6-AIPP-Fsk labels a 115-kDa protein that is inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B (Fig. 3A, position of cyclase indicated by the arrow). 125I-6-AIPP-Fsk also labeled three other proteins of 60, 33, and 18 kDa; however, the labeling of these proteins is unaffected by any of the drugs added for competition, and is therefore nonspecific.
Explanation:
meninges are brain structure that protect and cover the brain.
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