July 14th, 2007

science

Well that's... curious.

There is nothing I hate more than sacrificing a beautiful Saturday to a protein prep and then having it not work for mysterious reasons.

On that note, does anyone know why a His-tagged protein might suddenly not want to stick to a freshly charged Ni(2+) column? There seems to be a normal level of the protein in the "load" sample, I used appropriate concentrations of imidazole in the loading and elution buffers (20mM and 100-400mM, respectively), and I'm not even dreaming about approaching the column's theoretical binding capacity (12mg). So why the hell is all my protein in the flowthrough? If you have any ideas please share; I still have the FT and could potentially salvage the prep tomorrow. Though I don't really want to come in on Sunday as well. :P
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