Follow-up and another question

[Michelle Gleeson]

Hi Jeffery,

I can only answer one of your questions, but yes, imidazole does affect 
the Bradford Assay.  Imidazole is essentially histidine without the 
side chain, when you look at the structure.  I use 250mM to elute from 
my column (not especially high), and I notice an effect on my 
readings.  You can always include a imidazole blank at your 
concentration to correct for it.

cheers, 
Michelle Gleeson, PhD
Molecular Parasitology Unit
University of Technology, Sydney AUSTRALIA

----- Original Message -----
From: jhaines at uoguelph.ca (Jeffery Haines)
Date: Saturday, June 30, 2001 12:12 pm
Subject: Follow-up and another question

> Hello all,
> 
> Thank-you for all of the previous responses regarding the 
> precipitatioIncluding 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
> 
> 
> 

---