Question

1. Suppose that 50 μL of BSA-Cobalt salt solution was loaded onto a Sephadex G25 column, and eluted into three tubes. The amount of protein in each tube was quantified by the Bradford protein assay, and found to be as follows:

Fraction 1 contained 0 mg in 310 μL

Fraction 2 contained 10 mg in 290 μL

Fraction 3 contained 0 mg in 305 μL

What was the amount of BSA that was added to the column?

0.55 mg |
||

1.7 mg |
||

10 mg |
||

58 mg |
||

181 mg |

2. Suppose that a student used Sephadex chromatography to purify BSA (a protein) from 50 μL of a BSA-Cobalt salt solution. The total amount of protein collected from the column was determined using the Bradford assay to be 15.6 mg. What is the concentration of BSA is the original BSA-Cobalt salt solution? Give the final answer in mg/mL.

0.000312 (3.12 x 10 |
||

0.312 |
||

3.12 |
||

312 |
||

15 600 |

Answer #1

(1). The total amount of protein present(BSA) is equal to the sum of all proteins determined in fracction -1, fraction - 2 and fraction - 3 by Bradford protein assay.

Hence total amount of protein(BSA) = 0 + 10 mg + 0 = 10 mg (answer)

(2). Given the volume of BSA-Cobalt salt solution, V = 50
L
= 50x10^{-6} L x (1000 mg / 1L) = 0.050 mL

The total amount of protein collected using Bradford assay = 15.6 mg

Hence concentration of BSA is the original BSA-Cobalt salt solution = mass(mg) / V(mL)

= 15.6 mg / 0.050 mL = 312 mg / mL (answer)

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