2 replies to this topic

[Polymerase9]

Dear all

I have used affinity chromatography to purify a protein of my interest. After elution when i checked for the protein in SDS PAGE after silver staining I could see the desired protein band. But along with it I also see bands of molecular weight around 66kDa and another around 60kDa. I am sure that it is not a protein band and the gel was first stained with colloidal coomassie followed by silver. Please help me out to solve this problem.

Thanks in adavance.

[T C]

Hey,

How do you know its not a protein band?
Most of my proteins copurify with this 66 kDa band and once one of my seniors sequenced it and found it to be a chaperone.  

Besides, I get rid of this band at about 100- 150 mM NaCl conc on the FPLC, but not in case of all proteins.  Also, using 500 mM NaCl in wash buffer during affinity chromatography helps.

Best,
TC

Polymerase9, on Mar 24 2009, 07:28 PM, said:

Dear all

I have used affinity chromatography to purify a protein of my interest. After elution when i checked for the protein in SDS PAGE after silver staining I could see the desired protein band. But along with it I also see bands of molecular weight around 66kDa and another around 60kDa. I am sure that it is not a protein band and the gel was first stained with colloidal coomassie followed by silver. Please help me out to solve this problem.

Thanks in adavance.

[mdfenko]

then again, it may be an artifact that appears to be caused by dust and reducing agent.