Question

I am trying to purify DNA polymerase from cells DNA polymerase is a large soluble posivitely charged protein for each purification step below select which fractions you will keep in order to end up with pure DNA polymersase :

1. Cell fractioniation by centrifiguation:

a) Keep Nuclear Pellet

b) Keep mitochondrial Pellet

c) Keep microsomal supernatant

d) Keep microsomal Pellet

2) Salt fractionation with low salt concentration (low salt):

a) Keep precipitate

b) Keep supernatant

3) Gel Filtration

a) Keep later fractions

b) Keep middle fractions

c) Keep earlier fractions

4) Cation exchanger:

a) Keep middle fractions

b) Keep later fractions
c) Keep earlier fractions

5) DNA containing affinity column:

a) Later fractions

b) Earlier fractions

Answer 1

1. Cell fractionation by centrifugation technique separates different cell organelles from each other so we will keep A) nuclear pellet: where we will get the polymerase enzyme.

2.  Salt fractionation with low salt concentration (low salt) = low salt concentration will help the polymerase to get solubilize so the polymerase enzyme will be in B) solubilize fraction

3. Gel Filtration is the size exclusion chromatography in which the large protein will elute out first as it will not enter into the pores of the gel. So we will keep the c) Keep earlier fractions.

4. Cation exchanger: This involves negatively charged resins which will have affinity towards the positively charged molecules, since our protein DNA polymerase has positive charge it will have affinity towards the resins and therefore it will elute out in the later fractions so B) Keep later fractions.

5. DNA containing affinity column: this column will have affinity towards the DNA polymerase therefore while elution the later fraction will have the enzyme so we will keep A) later fraction.