A broad and strong A280 peak in a HIC chromatogram

Richard Van Frank vanfrank at iquest.net
Sat Feb 1 12:07:33 EST 1997


In article <mhsieh.2.32F2CAB7 at pop.service.ohio-state.edu>,
   mhsieh at pop.service.ohio-state.edu (Ming-Ching Hsieh) wrote:
>Hi Dear Netters:
>This really confused me. I used a HIC (Butyl Sepharose 4 Fast Flow from 
>Pharmacia) as the last purification step (preceded by DEAE and CM Sephadex), 
>and I observed a broad and strong A280 peak (from fraction #7 to #31, 2ml 
>each) in passed and washed fractions. The absorbance of 280 nm reached 2.499, 
>the limit of the spectrophotometer. I ran a SDS-PAGE and did not see any 
>protein bands by silver stain. In addition, I measured the protein 
>concentration by Bradford reagent and it did not show very very strong 
>reaction, either. Although it did not bother the protein I wanted which was 
>eluted by a negative linear gradient,  it did confuse me for the discussion. I 
>don't think I've got this high amount of proteins after two ion exchanges, and 
>I did not see this strong peak in those two columns, either. Plus, ammonium 
>sulfate (2.4M) should not interfere the 280 nm readings. What else?? Any 
>reasonable discussion is welcome. Thanks a lot in advance.
>
>Ming
>
What is the protein? Can you assay for activity or ? to determine if it is 
actually present? Try a dummy run of the coulumn, same buffer the sample is in 
but no sample, and elute it by your usual method to see if the peak you see is 
in some how reagent related. Under some conditions certain detergents will cause 
what you have described.
RMVF



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