Cleared lysate procedure

Remove 10 pL of crude lysate (spotted on the membrane as total protein) and remove the insoluble material by centrifugation. The supernatant (cleared lysate) and crude lysate are then used in the dot blot procedure described below to determine the amount of soluble and total recombinant protein, respectively. 

Nucleotidase activity was measured in human erythrocyte lysates which had been dialysed overnight against Tris/HCl buffer, pH 8.0 containing 20 mM mercaptoethanol and then diluted 1 5 in the same buffer. The assay mixture contained 0.5 ml haemolysate, 0.2 ml 0.1 M MgCl and 0.2 ml 0.04 M 5 ribonucleotide substrate in 0.05 M Tris/HCl buffer pH 8.0 and was incubated at 37°C for 1-2 hrs before adding 0.5 ml 20% trichloroacetic acid. Phosphate determination was carried out on the clear supernatant using the Chen modification (6) of the Fiske-SubbaRow procedure. 

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