I have to purify a bacterial protein from E. coli. My protein's yield is quite low. I have read that batch binding gives better yield, if so, for how much time do I have to incubate my protein with the resin? What are the factors which determine this time for batch binding of a protein.
Yeah, I mean, the first question is what % of your protein is in the soluble supernatant fraction. The second question is, what % of that are you recovering by the current purification regime and, what is the current regime. There may be many option to improve results.
Half and hour at 4 C is enough. Nonetheless batch adsorption only works when your protein takes time to reach the immobilized ions. If the his-tag is not available batch or column adsorption will not work.
Thanks for your suggestions.
Dear Martin, The pI of the protein is low and I use a higher pH buffer for solubility and proper folding. The problem is neither with precipitation nor improper folding. The expression of the protein is very low. We have optimized the conditions of highest possible expression, yet as compared to other proteins, the yield is quite low. I use Ni-NTA column followed by ion exchange. The His tag is well expressed and shows proper binding. To try a new method of binding and to get a bit better yield I wanted to do batch binding. So, I wanted to know the generalized time kept for protein binding to the resin.
Dear Omar Gonzalez-Ortega, if we incubate for some more time will there be an increased chance of non-specific binding? Suppose if I keep it for 1-2 hours at 4 degree can I ensure the maximal binding of my protein with a pay-off of some non-specific binding?
Non-specific binding with imac systems will always occur. It is not a matter of mass transfer limitations but a matter of pseudo-recognition.
There are many ways to skin this cat, and it is difficult to give excellent advice without combing through the procedure step by step. The non-specific binding may come as much from cell parts binding the resin as binding to the protein of interest itself. If you are trying to obtain a single pure protein, then consider doing a time course to extend the incubation time for as long as required to obtain sufficient yield (it will plateau - yes, there may be a trade off with noise accumulation, but, it depends). Then, wash the resin with high salt (up to 0.5M -- other regimes are possible of course, the point here is, a stringent regime) to help wash away non-specific binding - as well as washes with low concentrations of imidazole to eliminate weak pseudo-ligands. Finally elute in high imidazole, and carry on to next step. Good luck.
Your protein is His tagged. Does the plasmid contain T7 promoter? Change some modification in temperature, OD, IPTG concentration. If you find better expression then go for purification. The Talon metal Affinity resin from ClonTech works nice. Please share the temperature (starting from O/N preculture to subculture), IPTG conc and OD you had used.
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