I prepared a buffer containing the following: 50mM sodium phosphate, 250mM imidazole, 3M NaCl and 50% glycerol. When I autoclaved them all together, the solution turned yellowish. Is it still okay to use? I haven't done this before. (The product manual for the resin says that these concentrations with the glycerol should work just fine... but I'm not sure.) Thanks!
The following website quotes the following about imidazole-containing solutions: http://www.applichem.com/en/shop/product-detail/as/imidazol-pufferqualitaet/
''Solutions of imidazole are stable for several years ( at least 6 years). It is not necessary to autoclave the solution and to store it protected from light. Buffers containing imidazole will turn to yellow. These solutions are still suitable to elute His-tag proteins''
The glycerol when autoclaved, could turn yellowish brown. I would suggest leaving out the glycerol from the elution buffer and adding it later on. But as such the solution should be fine to use.
Any imidazole solution will turn yellow after a long-term storage in the fridge. This could already happen after autoclave if you use a low-quality powder.
The following website quotes the following about imidazole-containing solutions: http://www.applichem.com/en/shop/product-detail/as/imidazol-pufferqualitaet/
''Solutions of imidazole are stable for several years ( at least 6 years). It is not necessary to autoclave the solution and to store it protected from light. Buffers containing imidazole will turn to yellow. These solutions are still suitable to elute His-tag proteins''
By the way, why do you autoclave buffers for purification? Is it specific to the resin you are using? It is usually not necessary. Filtering using a 0.45uM membrane followed by degassing is generally considered as a good preparation.
Dont autoclave your buffers. If you use them for standard protein purifications, this step is not necessary. What you can do, to filter out precipitates or even microbes is to sterile filter with 0,2um filter. This will keep your buffer in a good state for a long time. Additionally, store the buffer at 4 Celsius Degrees. If you do that, your buffer will be free of clutter and microbial growth. If this still does not convince you, you can always finish up be adding Sodium azide. Sodium azide is an ionic solid. This chemical is used routinely in labs as preservative and biocide and helps to prevent bacterial growth over long term storage.
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