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After dissolving the biotinylated tracer in enzyme immunological assay buffer, the solution was used within a week or was alternatively frozen in smaller aliquots until use.
After addition of 1 ml of phosphate buffer, the solution was vortexed for 1 min and centrifuged at 4000 rpm for 10 min.
The gel was stained with 0.125% Coomassie blue at RT for 1 hr and destained with fixing buffer; the solution was changed every 15 min until caseinolytic bands were visible.
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After washing with NTMT buffer and PBSS, the solution was exchanged to 100% ethanol in two steps (50, 100%), and the samples were de-coloured for 1 h.
Next, 300 μL of lysis buffer was added, the solution was centrifuged at 15,000 rpm (Vision scientific Co., Korea) for 10 min and the supernatants were collected.
The pH of the solution was buffered to pH 7.2.
The pH of the solution was buffered with CaCO3 (0.25 mM) to pH 6-6.5.
The pH of the solution was buffered with CaCO3 (2.5 mM) to 7-7.3.
The solution was buffered with Tris/HCl to pH 7.4 at 37°C.
Various concentrations of cis-UCA were added to HBSS buffer, the solutions were adjusted to pH 6.5 or 7.4 and 300 μl of each solution was mixed with 15 μl of the BCECF-labelled cell suspension.
The pH of the buffer solution was 4.8 and the measured pH in the hydrolysis suspension was 4.53.
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