Indicate which type of chromatography can be used to separate or purify the foll
ID: 823947 • Letter: I
Question
Indicate which type of chromatography can be used to separate or purify the following protein mixtures and why?
a. Purification of Lysozyme (PI= 9.8, MW=14.3kD) from cell lysate at pH of 6.5 (hint: look at the protein charge) b. Purification of HA (hemagglutinin) tagged-GFP (Green fluorescent protein, MW= 27kD, PI=6.2) from cell lysate. c. Separation of Hemoglobin A (PI=7.1, MW=65kD), Hemoglobin C (PI= 7.5, MW= 65kD), Human carbonic anhydrase (PI= 6.5, MW= 28kD) and Bovine Carbonic anhydrase (PI= 6.0, MW=28kD) d. Purification of DNA from cell lysate
Will rate high if it is correct . Thanks!
Explanation / Answer
A. pH < pI of protein hence the protein will be having Positive charge.So v can use Cation ion exchange chromatography
B.HA-Tag is there.So Antibody against HA can be bound on to the column and can be used as Affinity chromatography.
C.First do gel filtration to seperate Hemoglobins and Anhydrases
then for seperating Hemoglobin A and Hemoglobin C and seperating Anhydrases use pH gradient chromatography called "Chromatofocussing"
D.DNA is negatively charged so v can use DEAE Anion exchange chromatography and elute at pH10.5.