Follow-up and another question

Jeffery Haines jhaines at uoguelph.ca
Fri Jun 29 21:12:34 EST 2001


Hello all,

Thank-you for all of the previous responses regarding the 
precipitation/aggregation of my hexahistidine tagged membrane protein.  
Including detergents like Triton-X100 (and other ones listed) initially 
seems okay, but I'm concered about having the detergent removed due to 
the fact that CD (circular dichroism) studies and lipid aggregation 
assays are being performed.    To all the posters who questioned what I 
am eluting with -- it is an elution buffer containing 6M urea and high 
conc. imidazole (sorry can't give exact amnt, I am at home writing this, 
lab book is sitting on desk at work).  If anyone has any new suggestions 
for preventing aggregation I would greatly appreciate it!   Did someone 
mention about using EDTA to elute protein from the column? -- would this 
help with preventing aggregation and wouldn't it strip the Ni?   Has 
anyone tried/had luck with these "Non Detergent Sulfobetaines" that the 
one poster suggested earlier?

My three *SHORT* but NEW questions are:
1)  Does imidazole (high concs. of it) affect the Bradford assay (ie. I'm 
wondering if I can check the protein concentration of fractions collected 
directly from the column so I can have a rough estimate of how much protein 
I'm losing upon aggregation).

2)  Has anyone ever tried using a double affinity tag on a protein 
before?  ie.  GST fusion and hexahistidine  -- any luck?/protocols?/pitfalls?

3)  Is there a website with a list of other affinity tags that can be 
linked onto proteins, or are GST and his tags the most common?


Thanks as usual to everyone for responses!
Jeff Haines
jhaines at uoguelph.ca





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