The absorbance at 340nm indicates that my protein binds to NADH or NADPH but how would I confirm that? I haven't got access to mass spec at the moment but was thinking of using a nucleotide HPLC column to qualify the 340 absorbance as NADH or NADPH. Is there anything else that absorbs at 34 that I may be missing? This thing remains tightly associated following two rounds of purification. My protein uses NADPH as a co-factor but I'm not aware of many proteins that tightly bind to NADPH. Any advice would be greatly appreciated guys!
Thanks in advance
Harry
Try using an assay that uses NADH or NADPH (don't add them to your assay) and has a higher binding capacity. This will tell you whether they are present.
Extract the protein with 1/2 volume of ice-cold 1.2 M perchloric acid. Neutralize the extract with 1/4 the original sample volume of 1.3 M potassium carbonate. Spin down the resulting potassium perchlorate in a microcentrifuge. Use the supernatant for HPLC nucleotide analysis.
Dear Harry,
You've done two rounds of purification - so I would think you're ok - but just be careful. Absorbance at 340nm is associated with protein aggregation. You probably don't want to be loading aggregates onto an HPLC.
Good luck,
James
Hey James,
Thanks for your answer. I wasn't aware of that. from the spectrum the peak at 280 and at 340 seem fairly sharp. It doesn't seem to be the profile of an aggregate.
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