Question

Data for a protein purification is shown. The protein has a characteristic absorption maxima at 557 nm. If I measure the OD557 for each purification step, MOST LIKELY, which step would show the least OD? Which would show the highest? Would they all be the same? Explain your answer.

Data for a protein purification is shown. The protein has a characteristic Total Activity (Units) 10.101 absorption maxima at 557 nm. If I measure the OD557 for each purification step, MOST LIKELY, which step would show the least OD? Which would show the highest? Would they all be the same? Explain your answer. Cell lysate Gel fitration Cation exchanger Affinity Total Volume (mL5 1 10,088 10,000 10,110

Answer 1

a) Cell lysate will have least OD.

b) Eluted sample after affinity chromatography will have highest OD.

c) No, OD will not be same for all of them.

d)

When you will start isolating your protein of interest from crude cell lysate, there will be lots of other proteins along with it, so the effective concentration of your protein of interest will be very less.

From the Beer - Lambert's law we know that absorption or the OD is directly proportional to the concentration for a certain wavelength. As the concentration is less in the cell lysate, OD will be less too for that specific wavelength.

Now, when you are purifying the protein by the property like charge, mass, affinity you will get rid of other proteins. So, the effective concentration of your protein of interest will increase. As the concentration increases, the OD or absorption will increase too.

If you look at the table provided, the activity of the protein is almost same after each of the purification test, but the volume of the protein solution used are decreasing. That means, after every step the concentration of your protein of interest is increasing, so as the OD will increase after every step.

Cell lysate has lowest concentration of the protein of interest, so OD is less. Eluted protein after affinity chromatography has highest concentration of the protein of interest, so OD is high.