urea/SDS, guanidine/SDS PAGE

[Curt Ashendel]

On 26 Oct 1994 23:04:08 -0400, 
HSchreiner  <hschreiner at aol.com> wrote:

>Is there a standard protocol to follow when running protein samples that
>have been solubilized in 6M guanidine hydrochloride (Gdn-HCL) on
>SDS-polyacryamide gels?  Are urea/SDS polyacryamide gels ever used?  What
>about Gdn-HCL/SDS-polyacrylamide gels?  Also, how does one go about
>treating the protein sample in Gdn-HCL prior to loading on to the ge?  Any
>suggestions would be appreciated.   Paul  (GonchaPa at umdnj.edu)
>
The ionic strength of samples in  6M Gu (Guanidine) will provide a great 
deal of conductivity and largely short out your electrophetic field.   
There are three solutions to this:

A.  Change your denaturant to urea or SDS by diaylsis prior to loading on 
the gel.  Complications include potential lack of solubility of the protein 
in the alternative denaturants and sample loss.  Also, it is a pain to do 
for a lot of small samples.

B.  Load identical amounts of identically denatured sample (including 
blanks, if needed) in every lane of the gel.  This helps to keep the 
ion front even.  Run the gel at a very low voltage/current, since it will 
generate a lot of heat at normal voltages (its resistance is very low).    
Be aware that the gel will run very slowly compared to normal.  [I have 
not tried this with 6M Gu, so I don't know if it will work, but it works 
with 0.5 M NaCl in the samples.] 

C.  Use a very, very small amount of sample per lane, minimizing the effect 
of the guanidine on how the gel runs.  You may need to keep it to as little 
as 1 to 5 ul, so one way to do this is dilute your 6M Gu sample into normal 
SDS loading buffer. Take the precautions mentioned in B.

In answer to the question about urea SDS gels, I think that they have been 
used on occasion, but the need for urea in addition to SDS  in the gel is 
not that common, as SDS adequately solublizes most proteins.  One exception 
I am aware of is extensively dye-modified proteins (as in "Rainbow markers" 
Tm of Amersham).  So it is unlikely that you will need urea in the gel.

Because of its affect on the electrophoresis process, Gu can not be 
included in standard SDS gels at concentrations where it is an effective 
denaturant.


I hope this helps.


Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu