Question

Can someone help me with this with explaination

14. You are studying a DNA damage pathway, have the following proteins in a mixture: Protein Molecular Weight (Mw) Isoelectric Point (pl) Chk1 53 kDa 8.7 MDM2 54 kDa 4.3 Intein-CDK2 55 kDa 8.8 GST-p53 68 kDa 6.0 His-Cyclin E 70 kDa 6.0 GST-GADD45A 95 kDa GST-ATM 375 kDa 6.3 5.9 Which type(s) of chromatography would you use to purify each of the following proteins from this sample? Include the eluting agent (such as NaCl) and pH of the buffer if relevant. a. ATM: b. Cyclin E: C. MDM2: d. CDK2 e. GADD454 f. p53: Elution Buffer/pH: Elution Buffer/pH: Elution Buffer/pH: Elution Buffer/pH: Column 1: Column 1: Column 1: Column 1: Column 1: Column 1: Column 2: Column 1: Column 2: Elution Buffer/pH: Elution Buffer/pH: Elution Buffer/pH: Elution Buffer/pH: Keep Elution or Flowthrough: _ g. Chk1:

Answer 1

A) size exclusion chromatography with pH 7.0 as in this chromatography large molecules will come 1st as it does not enter into beads so elute early so ATM will come first.

B) Nickel tag affinity chromatography. This chromatography have specific beads coated with Ni2+ as nickel have high affinity for histidine amino acid so it will specifically His tag cyclin E

C) Anion exchangers with pH 5.0, this chromatography will bind to negative charged molecules as beads having positive charge. Now at pH 5.0, only mdm will be negative charge as pH 5 is above than its pI value. Any protein above it's pI value is negative and below its pI value is positive.

D) Anion exchanger with pH 8.8

E) Size exclusion chromatography where in 2 elution fraction this specific protein will elute

F) Glutathione Sepherose bead chromatography. This chromatography having glutathione beads which binds to glutathione Sepherose tag proteins.

G) Chk specific antibody affinity chromatography or Anion exchanger at pH 8.7 but in this case there will be contamination of cdk2 protein

Hope it's clears . Thanks