Adventitious chemical binding
Posted 23 November 2009 - 10:34 PM
This is my first post and probably not the last. I have been working with this protein for quite some time and have been having trouble with activity assays. It is a soluble heme protein. The literature purification procedure (following ecoli lysis +centrifugation) involves ammonium sulfate fractionation, buffer exchange into 50 mM tris, anion exchange chromatography (0-500 mM NaCl) and completed with size exclusion in 50 mM tris (pH 7.5). (unfortunately a his-tag could not be added) The literature says that it is pure (as does the gel), however, the UV/visible spectrum varies somewhat from batch-to-batch as does the EPR signature. The activity results are also variable. I perform the purification very rapidly so I don't think there is an issue of protein degradation occuring (although I may be wrong). I have an inkling that there might be adventitious binding of unknown chemicals in either the active site or other regions that may be affecting the activity. I was wondering if this is a common occurence, or if there is something I'm missing altogether.
Any feedback or questions on this issue would be greatly appreciated.
I just want the damn protein to work
Thanks in advance
Posted 24 November 2009 - 07:27 AM
when determining purity on the gel, how much do you load? with what do you stain (coomassie or silver)?
you may find variable contaminants in your preps.
as for other chemicals, do you use the same solutions for each prep? same stocks? same cleaning procedure?
if so, then there should be the same chemical complement in all preps. if not then maybe.
genius does what it must
i do what i get paid to do
Posted 24 November 2009 - 07:51 AM
If you think that this maybe a problem, what you would want to consider next is a way to determine what fraction of your preparation is active. For example, if you have a heme protein, lets say that your active form binds heme with a 1:1 stoichiometry (and your inactive form does not bind heme). You can measure the amount of heme in your prep and if you know that active protein binds heme and the denatured form does not, you can calculate the amount of active protein.