Hi,
Can ATP bind to Q column at pH=7.5 and be eluted by NaCl salt gradient? And can ATP be detected at 280 nm?
I washed my Q column with the binding buffer containing 5mM ATP/3mM MgCl2 to get rid of DnaK and I have a massive peak at 280nm (that do not contain proteins as seen on SDS-PAGE) at the beginning of the elution on FPLC.
Thanks for your help.
N
ATP has an absorbance peak at 259 nm (extinction coefficient is 15,400 M-1cm-1), and has substantial absorbance at 280 nm. It will bind to a Q column if the salt concentration is low enough because of its negatively charges phosphates, and will be eluted by low concentrations of salt. So I think you are right that the large absorbance peak is ATP.
Thank you Adam for your answer. The salt concentration was about 50 mM which is very low...
If you are attempting to purify a protein and this is interfering then you may want to either dialyze (30 kD exclusion) or desalt (Buffer Exchange) with G25 - assuming since the equipment is fast flow chromatography.
Separation of nucleotides by anion exchange chromatography is not only possible, but a standard technique for example to measure ATP/[3H]ADP exchange reactions. I used a gradient of 0-100 mM KCl in 20 mM TEA on a 1 ml MonoQ column.
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