I need help with B!! 5. You have prepared 430 mL of crude homogenate by homogeni

Question

I need help with B!!

5. You have prepared 430 mL of crude homogenate by homogenizing 250 grams of chicken liver with 430 mL of extraction buffer.

a. 25 L of the Crude was added to 1,975 L of buffer and the A280 was observed to be 0.52 while the A260 was 0.88. What was the Crude’s protein concentration and how many mg of protein were in the Crude?

ANSWER:

C=0.1372 mg/mL

Crude concentration= 4.72 g

b. 50 L of Crude was added to 450 L of buffer and 5 L of this material was assayed for LDH activity. The A340/sec was 0.068.

How many units of LDH were in the Crude? What is the Specific Activity of LDH in the Crude Chicken Liver Homogenate?

Explanation / Answer

According to the question

As we know that

Based on the given information:

Amount of chicken liver used = 250 grams

Volume of extraction buffer used = 430ml

Then,

250grams chicken liver/430ml extraction buffer = 0.581g chicken liver per ml extraction buffera

Then,

a) 25ul Crude + 1975ul buffer, A280 = 0.52 and A260 = 0.88, dilution factor = 2000/25 = 80x

Cruude's protein concentration

Protein concentration can be determined using the formula:1.55*A280 - 0.76*A260

So,

=1.55*0.52 - 0.76*0.88 = 0.1372mg/ml

Then,

Protein concentration in Crude = Protein concentration *df *430= 0.1372 * 80 *430 = 4719.7mg or 4.72g

Therefore,

Crude sample contains 4.72g of Protein.

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