In the LDH activity assay Sample B gave 03 Amin rate when us

In the LDH activity assay, Sample B gave 0.3 A/min rate when using a 1:50 dilution, 10 µL of the dilution was added to the reaction which had a final volume of 1.21 mL. After the activity assay, you performed a BCA assay to measure the total protein in Sample B. When 25 µL of 1:100 dilution of Sample B was used in the BCA assay, from the standard curve you calculated the protein concentration of the dilution was 200 µg/mL. Please answer following questions and show your work, and carry proper units during your calculation.

a. What is the activity of Sample B?

b. what is the total protein concentration of Sample B?

c. what is the LDH specific activity of Sample?

Solution

10 µL of the dilution was added to the reaction which had a final volume of 1.21 mL

Use the formula N1 V1 = N2 V2. Dilution is 1:50. This means that initial stock was 50X which was diluted to final concentration = N1 = 1X

1*10 = N2*1.21; N2 = (10/1.21) = Answer to Part \'a\'.

b

Given: 25 µL of 1:100 dilution of Sample B was used in the BCA assay from the standard curve you calculated the protein concentration of the dilution was 200µg/ml.

This means that the stock solution was 100X and working solution was 1X.

Protein concentration in working solution is 200µg/ml. So, protein concentration of stock solution will be 200*100 = 20,000 µg/mL or 20mg/ml

c

Specific activity is micromoles of substrate utilized (BCA) or product formed per ml of enzyme (LDH) per unit time.

20 *1000 µg/mL * 0.3/ molecular weight of BCA (in µg)

From internet, molecular weight of BCA= 213.3gm = 213.3 * 10⁶µg

So, specific activity = 6000/213.3 * 10⁶ µM/ml/min

= 0.0281 *10^-3 µM/ml/min