Stephen P. Driska PhD wrote:
>>newera at plaza.snu.ac.kr wrote:
> : Can coomassie blue cause an artifact of proline content in
> amino acid analysis
> : of a protein?
>> : I have amino-acid-anayzed my protein 3 times, which is transfered on
> : PVDF membrane and stained with coomassie blue. Each time, I have got
> : a different result in terms of proline content but the contents of
> : the other amino acids have been measured almost the same.
>> : What is the possible causes of this problem?
>> : --
> : email : newera at plaza.snu.ac.kr> : address :
> : Lee, Ji Hyun
> : Laboratory of Physical Pharmacy(Prof. Lee, Bong Jin)
>> : Seoul National University
> : College of Pharmacy
> : Shinlim-Dong, Kwanak-Gu
> : Seoul 151-742, Korea.
>> It's an obvious thing, but could you try removing the coomassie blue
> from the PVDF? Also, it might help people suggest an answer if you said what
> type of machine or column was being used for the analysis. A few years ago
> we were having a problem with coomassie blue and the Waters PicoTag system,
> but we had enough protein that we could remove the blue dye with ethanol, I
> think 90%. But the protein was freeze dried on the walls of a sample tube,
> not stuck to a PVDF membrane.
>> Good luck with your project
> - Steve
>> --
> Steve Driska, Physiology Department, Temple University Medical School
> Philadelphia, PA 19140 USA (215) 707-3283
>driska at astro.ocis.temple.edu
Check the quality and age of your chemicals. When I did my PhD-thesis,
too many years ago, I had similar problems with our protein sequencer.
After changing to a new Coomassie and SDS batch of higher quality, the
problems disappeared.
If you do not want to buy better Coomassie, check Anal. Biochem. during
the last two, three years, or so; I have seen at least two papers about
prpearing higher quality Coomassie from usual commercial products.
Achim
