I am doing some his-tagged protein purification and I would like to see if the chromatography works by running a standard. Is there a cheap histidine tagged protein people tend to use?
Also the imidazole I use is quite dirty and absorbs at 280nm. Please suggest a product that does not have absorption at this wavelength if you know one.
You may use bradford to estimate protein concentration.
https://www.thermofisher.com/order/catalog/product/LC5606
Dear Pavel
Sincerelly i never used a specific standard to set up the imac because recombinant proteins are quite expensive but i used a clone present in the lab that i know that work well.
Addgene ( https://www.addgene.org/) is a large repository of plasmid. they share it for less than 100 euro. i suggest to you to find a good positive control as a gfp, thioredoxin his tagged and produce it by your self.
Regarding imidazole, the bioultra purity level of Sigma has low absorbance at 280 nm and can work for your purpose https://www.sigmaaldrich.com/catalog/DataSheetPage.do?brandKey=SIGMA&symbol=56749
good luck
Manuele
Long ago, as a student, I had a similar problem, as we did not have the funds for ultrapure guanidine HCl. I cleaned up a dirty version by adding activated charcoal to the solution and filtering it first over filter paper, then through membrane filters. Worked perfectly.
Maybe this is something you could try with imidazole, if your lab cannot afford the ultrapure reagent.
But I warn you, it will be dirty work - wear an old labcoat !
Dear Pavel: I don't know, if that's helpful for you, but we're offering a his-tagged GFP protein in quantities of 50 µg and 250 µg. It is quantified via 488 nm wavelength and can be used as a standard.
Hope that helps, Roland
https://cube-biotech.com/products/plastic-accessories/green-fluorescent-protein-egfp/159/egfp-protein-his-tag?number=29903&c=231
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