You run LDH assay on one of your samples, and obtain activity of 1.5 A/min. You

Question

You run LDH assay on one of your samples, and obtain activity of 1.5 A/min. You then dilute the sample 1:10 and obtain activity of 1.2 A/min... (Please answer as many of the following questions that don’t require more data.. 2,4,5,6) Prelab Questions #2C 1. What reagents (and how much of each) do you need to add to the cuvette when running the LDH enzyme assay? 2. You run an LDH assay on one of your samples, and obtain an activity of 1.5 AA min. You then dilute the sample 1:10, and obtain an activity of 1.2 Amin. Is this the activity that you would expect from a 1:10 dilution of your original solution? Why? What is happening in these assays 3. Which samples do you need to assay for LDH activity? 4. Why do you need to set the spectrophotometer for 340 nm when running the LDH assay? 5. You obtain a rate of 0.8 AA/min from one of your samples (Sample A). You added 10 al of a 1:10 dilution of Sample A to a total volume of 1.21 ml. What is the corresponding activity (in ol/min/ml in the original sample (Hint: you may wish to consult the Calculation Hints page for assistance in calculating this value.) 6. Sample B gave 0.3 A/min when using a 1:50 dilution in the same assay. What is the corresponding activity (in ol min/ml) in the original sample? which sample (A or B) has the higher activity?

Explanation / Answer

2. LDH assay or Lactate dehydrogenase assay include reactant as NADH and NAD+ as a product. As activity decreases on performing the dilution because it is required to dilute the enzyme before assay because we want to make sure that diluted sample should have an absorbance which lie between standard curve.

4. We need to set the spectrophotometer at 340 nm while running the assay because NADH has an absorbance visible at 340 nm.

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