Purification of connexins and autoradiography:

Step 21: the resulting pellet was resuspended in 30 mL 5 mmol/L Tris, pH 10 at room temperature and then homogenized (step 22). Step 23: after addition of 30 mL 0.6% N-laurylsarcosine in 5 mmol/L Tris (pH 10) and, step 24, 10 mins stirring at room temperature, the suspension was added (step 25) to a density gradient (20 mL test suspension, 8 mL 35% sucrose, 0.3% deoxycholate in 5 mmol/L Tris, pH 10; 5 mL44.5% sucrose, 0.3% deoxycholate in 5 mmol/L Tris, pH 10). Step 26: after 20 mins at room temperature the solution was centrifuged (65,000 g at 15°C for 60 mins) (step 27), the material of the 35% to 44.5% interface was collected and (step 28) resuspended in 45 mL 0.3% deoxycholate in 5 mmol/L Tris (pH 10).This was centrifuged again (three samples of 15 mL) in a density gradient (15 mL test volume, 9 mL 35% sucrose, 0.3% deoxycholate in 5 mmol/L Tris, pH 10; 9 mL 44.5% sucrose, 0.3% deoxycholate in 5 mmol/L Tris, pH 10) for 60 mins at 65,000 g at 15°C (steps 29, 30). Steps 28 to 30 were repeated (as steps 31, 32). The interface material was collected and diluted 1:1 in 0.3% deoxycholate in 5 mmol/L Tris, pH 10, and centrifuged at 106,000 g for 30 mins at 15°C (steps 33, 34). The pellet containing gap junctions with nonjunctional membranes was washed in 5 mmol/L Tris, pH 10, and centrifuged in a density gradient (test volume, 9 mL 31.5% sucrose, 0.3% deoxycholate in 5 mmol/L Tris, pH 10; 9 mL 35% sucrose, 0.3% deoxycholate in 5 mmol/L Tris, pH 10) at 65,000 g for 90 mins at 15°C (steps 35 to 38). Find out more about your chance to get Symbicort Price best price at best online pharmacy.

Category: Cardiology / Tags: Antiarrhythmic drugs, Antiarrhythmic peptides, Cardiac arrhythmia, Cellular coupling, Gap junction

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