Follow-up and another question
Michelle.Gleeson at uts.edu.au
Sun Jul 1 19:41:23 EST 2001
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.
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
> assays are being performed. To all the posters who questioned
> what I
> am eluting with -- it is an elution buffer containing 6M urea and
> conc. imidazole (sorry can't give exact amnt, I am at home writing
> lab book is sitting on desk at work). If anyone has any new
> for preventing aggregation I would greatly appreciate it! Did
> mention about using EDTA to elute protein from the column? --
> would this
> help with preventing aggregation and wouldn't it strip the Ni?
> 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
> 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
> 3) Is there a website with a list of other affinity tags that can
> 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
More information about the Methods