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
