Guanidine samples on SDS PAGE

[Squid]

Is there some trick to this? When I add my SDS loading buffer to the sample
containing guanidine (6M) it produces large amounts of blue solid....I'm
sure I should have paid more attention in chemistry class!! My protein is
6XHis tagged, and must be solubilized in 6M guanidine HCl so that it can
bind the affinity column. I want to run the samples on a gel (quickly!!) to
monitor purification, and was hoping not to have to dialyze the guanidine
out first, which causes my protein to become insoluble again...

thanks

julie

[Michael L. Sullivan]

I think novagen might have some information about this on their web site.
You do have to get rid of the guanidine for SDS-PAGE, or at least reduce it
to some low amount-- not sure what.

An alternative is to use urea as your denaturant. This is compatible with
SDS-PAGE.

Mike

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

---

[Nick Theodorakis]

The guanidine is likely precipitating SDS, which is not terribly soluble in many
high-ionic strength solutions. Even if it was, the high ionic strength of 6M
guanide would be problematic for electrophoresis. You could try either dialyzing
against urea (which is neutral and is compatible with SDS) or using urea in the
first place instead of guanidine.

Nick

--
Nick Theodorakis
nicholas_t...@urmc.rochester.edu

[Christof Gaenzler]

Try a non denaturing gel and buffer, leave the SDS out. But with 6M GHCl
you won't get a nice gel.

--

---
Christof Gaenzler
Deutsches Krebsforschungszentrum (DKFZ)
Applied Tumorvirology (ATV)
F0200
INF 242 - 69120 Heidelberg
T: +49-6221-42-4937
F: +49-6221-42-521008
EMail: c.gae...@dkfz.de

[Mark Bowen]

SDS is not soluble in Gnd at high concntrations. you can dilute the samples if
the protein concentration is high enough. normally I use TCA precipitation from
my Gnd solution followed by acetone rinsing and resuspension in SDS buffer with
0.1M tris pH 8. this can be done on fairly small samples for PAGE.

Squid wrote:

--
Mark Bowen

Laboratory of Axel Brunger
Howard Hughes Medical Institute
Stanford University

lab (650) 736-1715
fax (650) 736-1961
mark....@stanford.edu

[Kevin Brady]

If you already have samples in GnHCL then you could buffer exchange down a PD-10 column (Pharmacia no aff.). Just equilibrate the column with your Urea buffer, add your sample to the top, add more urea buffer. Collect 1ml fractions, spec them for presence of protein and hey presto, your protein is still solubilised, but now in urea. Couple of points : 1) Your sample will be 1.5-2.5 times more dilute so you may need to concentrate (ultrafiltration etc) 2) Make urea solutions up fresh.
Cheers
Kevin

-- 
Dr Kevin Brady
Organic Chemistry Section
School of Chemistry
University of Nottingham
Nottingham
UK
NG7 2RD
#################################################
Email : Kevin...@nottingham.ac.uk or
        ke...@basil.chem.nott.ac.uk
URL   : http://www.nottingham.ac.uk/~pcxsc/thomas
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[Choe Woo-seok]

Try to precipitate your proteins by adding some buffer or simply water.
Then take the supernatant and precipitates separately and add gel loading
buffer containing
SDS to each part. Your precipitants will be dissolved without difficulty.
But other proteins
which is still soluble may not be detected on Gel due to dilution.

-------------------------------------------
Woo-seok Choe
Dept. of Chemical Engineering
University of Cambridge
Pembroke Street
Cambridge CB2 3RA, UK
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Phone : +44 01223 740 979 (Home)
+44 01223 334 786 (Office)
Fax : +44 01223 334 796
E-mail : ws...@cam.ac.uk
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