A broad and strong A280 peak in a HIC chromatogram

Leemor Joshua-Tor leemor at CSHL.ORG
Sat Feb 1 18:13:28 EST 1997

>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.

Do you have Triton in there? Triton has a peak at 277 nm.

  Leemor Joshua-Tor, Ph.D.
  Assistant Investigator
  Keck Structural Biology
  Cold Spring Harbor Laboratory           Tel. (516) 367 8821
  1 Bungtown Road                         Fax  (516) 367 8873
  Cold Spring Harbor, NY 11724            e-mail: leemor at cshl.org

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