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To the supernatant, 70 ml of buffer II (pH 7.5) (20 mM potassium phosphate, 100 mM KCl, 24 mM imidazole and protease inhibitor cocktail) was added.
Twenty microliters of 3%% ethanol iodine solution was dissolved in 10 ml of buffer.
60 mg of the dry hydrogels were placed in 25 mL of buffer solutions (1.2 and 7.4) at ambient temperature.
The column was regenerated with 17 ml of buffer B at 1 ml/min.
The column was then washed with the equivalent of 3 column volumes (20 ml) of buffer.
The resin was washed twice with 1 ml of buffer I.
Then, 0.5 ml of buffer containing 12 mM p-nitrophenylphosphate with or without ouabain were added to each sample.
Each pellet was re-suspended twice in 10 ml of buffer 2 and centrifuged (1,500×g, 15 min).
The lysate was added to 10 ml of buffer and centrifuged at 800× g for 15 minutes.
Similar(2)
Add equal volume (4 ml) of buffer-saturated phenol-chloroform, mix vigorously for one to two minutes.
Here, haemoglobin concentrations of ≥50, ≥75, ≥100, and ≥200 ng/ml of buffer solution were taken as cut-off values.
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