The interface material was centrifuged at 106,000 g for 30 mins at 15°C and washed, and the pellet containing the gap junction proteins was obtained (steps 40 to 42). For detection, either SDS-PAGE or PAGE was used.
SDS-PAGE: After the purification procedure, the pellet obtained from step 42 was separated by PAGE, as described above, but under nondenaturing conditions, ie, without SDS and mer-captoethanol. The stained gel was submitted to radio-fluorography. The gel was exposed to 50% trichloroacetic acid for fixation for 12 h and thereafter to dimethylsulphoxide (DMSO) for two 30 min periods, followed by exposure to 22.2% diphenyloxazole in DMSO for 3 h. Thereafter, the gel was washed with water for 2 h and dried (60°C, 90 mins). The gel was incubated for 60 days at -70°C in an x-ray film cassette with an x-ray film after being exposed to flashlight (20 W). PAGE: As a running gel, 12.5% (weight/volume) acryl-amide SDS-PAGE was used (prepared from a 30% acryl-amide, 1.0% bisacrylamide in water monomeric solution; 10% ammoniumpersulphate); as a stacking gel 5% (weight/volume) acrylamide SDS-PAGE was used (prepared from a 30% acrylamide, 1.0% bisacrylamide in water monomeric solution; 10% ammoniumpersulphate); the running gel buffer solution was 0.18 mol/L Tris, 0.1 mol/L glycine, 3 mmol/L SDS; the same solution was used as stacking gel buffer solution and as electrode buffer. Sample buffer was 20% SDS, 10% glycerine, 0.2% mercaptoethanol and 0.2% bromphenol blue in 0.25 mol/L Tris, pH 6.8. The peptide marker was a low molecular weight SDS kit from Sigma (St Louis, Missouri, USA). Commassie brilliant blue was used for staining. Thus, it was possible to verify that connexin 43 could be separated using the method described above. Your most trusted pharmacy offering Symbicort Dosage let’s go here and giving you very fast shipping.