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LDH Lab 3 Protocol - Section N TA Josh LDH Lab 3 Protocol...

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Section N TA: Josh LDH Lab 3 Protocol Materials: Piston pipets, test tubes, phosphate buffer, NADH, pyruvate, serological pipets, column fractions Procedure: 1) Reassay the fractions from last period that gave unacceptable absorbencies. Also make duplicate protein measurements for the elute fractions. 2) Absorbance is proportional to concentration, so adjust the concentrations of the unacceptable absorbencies to the appropriate concentrations. For example, if the absorbance was 0.1, to get into the appropriate range, 0.3- 0.8, you should dilute the fraction less next time, around 5-fold less to get an absorbance of 0.5. 3) Assay fractions 8-12 for protein a second time once measurements are appropriate. 4) Perform enzyme assays on the elute fractions and the fractions immediately before and after those that gave a + on the spot plate tests. 5) For these enzyme assay dilutions, start with a 10-fold dilution on those that gave a faint color, drawing 40 ul of enzyme with 360 ul of phosphate buffer. Keep column fractions on ice, but dilutions at room temp.
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