Protein purification problem

gilbe...@hotmail.com

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Mar 12, 2004, 4:14:44 PM3/12/04

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I am currently working on the most difficult project I have ever seen and have finally been forced to take the plunge and ask for outside assistance.

This protein is a novel type from bacteria. Size exclusion chromatography suggests it to be 35-45kDa. It does not stain by coomassie, silver stain or any traditional techniques on SDS-PAGE. However, it is stained by Stains-All, but this shows it to be stuck at the stacking/resolving gel interface.

We have tested for amino-sugars and found none.

Does anyone have any idea why a protein would not travel through a polyacrylamide gel at all?

If anyone has noticed anything like this before I would love to speak with you in more detail.

Yours sincerely,

Jack Gilbert

http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0

Dr. Hiranya S. Roychowdhury

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Mar 12, 2004, 10:36:50 PM3/12/04

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How do you know that what you are seeing with stains-all IS the protein of
your interest? I find it curious that a polypeptide will not stain with CBB-R
or silver! Stains-all stains phosphate and glycosidic residues and, to a
lesser extent non polar conjugates (albeit showing different colors). So, if
what is being stained does not migrate in an electric field, I doubt that it
is a polypeptide.

Quoting gilbe...@hotmail.com:

--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---

Sulakshana Mukherjee

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Mar 12, 2004, 11:02:24 PM3/12/04

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Hello!
Use Mass Spectroscopy to determine your protein's Mol. Wgt. This can
also give the purity of your sample. You can as well use capillary
electrophoresis for the purpose.

with best regards,
Sulakshana

****************************************
Sulakshana Mukherjee
Research scholar
Molecular Biophysics Group
Tata Institute of Fundamental Research
Homi Bhabha Road
Mumbai-400005
India
Phone: +91(22)22804545 ex-2271
****************************************

---

P.C.

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Mar 13, 2004, 1:45:20 PM3/13/04

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well, it does sound like it could be not a protein at all, I agree with
H.S.R. But assuming it is a protein, one thing that comes to mind is
boiling.

Some proteins do not tolerate boiling. One example I had - misterious
dissapearances of a recombinant viral capsid protein. I had tons and
tons of it, but somitimes I would see nothing just some weak ugly smears
upwards and some stuff in the stacking gel. The solution was to avoid
boiling and instead to leave it at room temp for 10-20 min in the SDS
sample buffer (it is enough to reduce cystein residues in most cases).

Peter

nanc...@gmail.com

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Mar 1, 2013, 12:36:32 AM3/1/13

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Please take a look at www.ehsystems.com It may help you. Some instruments are not put on the website as for now because either they are being tested before launch or in prototyping stage, we would still be happy to provide information through email.
na...@ehsystems.com
001 908 998 2858