Savita Visal wrote:
>> Dear Friends,
> I want to sequence protein of interest. I have my protein of interest as a
> single band on SDS_PAGE. I've dried this gel. I would like sequence this
> protein..the problem is elution from the SDS-PAGE. I have read so many
> protocols and at this stage I'm totally confused ..which protocol do I
> use? MW of my protein is 55kDa. Could anyone share their experiences and
> guide me to which is the best protocol I could use?
> Thanks
>> Savita
>4104vsav at vm1.ulaval.ca
Savita,
It seems there are a couple of things to be considered.
1) Do you want to sequence from the dry gel you have in hand right now?
If you do, How is it stained? You will probably have to destain it
before transferring to membrane. I Coomasie stain the membrane then.
The microsequencer we have in our department is an ABI machine and it
will sequence Coomasie stained bands, but only if it is stained with
G-250 coomasie, apparently R-250 will come off during sequencing and
interfere with the chemistry. So you need to consider that.
How was your gel run? There is some evidence that standard SDS-PAGE
procedures MAY result in artifactual N-terminal blockage. To avoid this
pre-run your gel with 0.3mM Nathioglycolate. I have done this with
success. If you don't take some precautions and you submit bands for
sequencing and the sequencing is unsuccessful you don't know if your
protein is naturally N-term. blocked or if its artifact so I take the
precautions.
As to blotting I use a semi-dry blotter, (Bio-Rads) and a Tris-Glycine
transfer buffer called Bjerrum and Schafer-Nielson Buffer w/ SDS. With
this equipment I can load an outside lane on my gel with pre-stained
SDS-PAGE standards and periodically during the transfer I can stop the
current and carefully peal up the edge and monitor the transfer of
protein of the approximate size. I use PVDF with the smallest pore size
I can find to avoid transferring through the membrane.
Hope this helps
Happy Hunting
--
>>>>>>--------------------------->
Ron
Tate
Lab of Franklin Leach
Dept. of Biochem. & Molecular Biology
Oklahoma State University
rtate at bmb-fs1.biochem.okstate.edu
(405) 744-9326
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