Dennis -
This doesn't sound hard. You could accomplish this with an HPLC method that:
1) Separates the two related proteins from each other, and;
2) Gets them to elute in discrete peaks separated from any other proteins.
I assume you have authentic standards you can use to determine the elution
position of the proteins in any particular mode of chromatography.
Separation of two proteins differing only in a couple of residues at the
C-terminus is easy. It's possible to resolve proteins this size based on a
sinlge residue difference if you use the right mode of chromatography (e.g.,
if the residue in question is hydrophobic, then try Hydrophobic Interaction
Chromatography [HIC]. If it's charged, then try ion-exchange chromatography,
etc.).
The following paper is instructive on this matter: Loayza et al., J.
Chromatogr. B, 662 (1994) 227-243. They were isolating fibrolase from
copperhead snake venom. Starting with crude venom, the activity was purified
through successive steps involving HIC, hydroxyapatite, anion-exchange, and
cation-exchange chromatography. A single peak of activity was obtained from
the first three steps, but three isoforms were resolved at the
cation-exchange step. That's the sort of discrimination you should be
looking for too; you don't want to separate your native and truncated
proteins until the last step.
It is unlikely that you'll need to use 4 complementary steps to isolate and
resolve your proteins. If you're lucky, two will do. If your sample
quantity is limited, then use 1.0- or 2.1-mm i.d. columns. Which columns to
try depends on such factors as the pI of your proteins and the sequence
that's missing from the C-terminus. You can contact us if you want to
discuss the details.
Best regards,
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD 21045 USA
tel: (410) 992-5400 FAX: (410) 730-8340
************************************
<< Subj: Misc - Question
Date: 05/15/2001 7:46:42 PM Eastern Daylight Time
From: dleveson@bccancer.bc.ca (Dennis Leveson-Gower)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
Hi everyone,
I have a problem and I thought I might be able to find some help in this
discussion group.
I am co-expressing two proteins in CHO cells and then trying to detect if
one or both of them are expressed. The problem is that the two proteins are
identical except for short peptide sequence at the C-terminus. I have
antibodies that react with both fragments but not with each individually.
The whole proteins should be approximately 50 and 53 kDa in size so
separation on Western would be difficult.
I need a way to take whole cells, break them open, and then see if both
proteins are being translated.
Thanks,
Dennis.
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