You have purified LDH using affinity chromatography. Using the Bradford assay, y

Question

You have purified LDH using affinity chromatography. Using the Bradford assay, you determined the total protein concentration of the column fraction to be 7.5 mg/ml, in a final volume of 2.6 ml. You diluted some of your column fraction to 1 mg/ml using SDS PAGE sample loading buffer, and loaded 15 µl on to an SDS PAGE gel. You also loaded 10 µl of a 1mg/ml LDH standard in an adjacent lane. The stained SDS-PAGE revealed that your LDH column fraction contained a number of bands besides LDH. Upon scanning, you found that the LDH band in your column fraction had an optical density of 42,000 pixels. The optical density of the LDH standard band was 58,000 pixels. a. What is the percentage purity of your LDH column fraction? b. How many total mg of LDH are there in your column fraction?

Explanation / Answer

C of LDH pure 1mg/l optical density of pure LDH = 58000 and the optical density of LDH in my column is 42.000 so

1mg/l x 42/58 = 0.724mgl LDH

b) in the sample we have 0.724 mg/l de LDH

C1 x V1 = C2 x V2 C1 = 7.5mg/ml V1 = 2.6ml V2 = ? C2 = 1mg/l =) V2 = C1xV1/C2 =  7.5mg/ml x 2.6ml / 1mg/l =) V2 = 19.5ml now return us to the column we have  0.724 mg/l de LDH

mg of LDH = 19.5ml x 0.724mg/l =) mg of LDH = 14.118mg of LDH

a)

C =14.118mg/2.6ml = 5.43mg/ml

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