HELP!!!!absorbance peak and radioactivity do not match??? - (Sep/16/2011 )
hi guys, i have been trying to isolate polyglutamal substrate from a enzyme reaction with e-coli FPGS+5-formal THF on a De52 column. I have run standards before just to check where the tri-di- and mon- glutamates would come out. However, everytime when i do the reaction, the absorbance peak and the counts peak ( in cpm, and i use H3_glutamates) do not match and the radioactivity count peak are always 1 or 2 fractions away from the OD peak. The most reliable information does come out from the counts since di-glutamate will give a strong count peak. I am absolutely confused and is out of ideas!!! would the buffer affect this, the buffer only contains tris, mecarptaethanol and sodium acetate and i run it on a gradient.
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how do you collect fractions?
volume between the uv monitor and the fraction collector?
to avoid fraction offset, we set a delay between drop count and fraction switch based on the volume between detection and drop formation.
i collect the fraction with a fraction collector, it is quite old, fraction volumes are around 2 ml( i collect 40 drops) then i use a t-can to read the absorbance at 300 nm .
and by delay, u mean?
since you are reading and counting the fractions, the delay is not an issue.
what you are probably seeing is the bulk of the reactants in the major peak and the product as either a shoulder or a minor peak or as part of the tailing of the reactants peak.
you may have to pool and concentrate the radioactive fractions and apply them to a different column type (eg- gel filtration) to further purify the product.