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Methods Digest, Vol 19,Issue 1-younes sadramehr SDS-PAGE repurification

Methods Digest, Vol 19,Issue 1-younes sadramehr SDS-PAGE repurification - Protocols and Methods Forum

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Old 12-09-2006, 06:29 AM
Jess
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Default Methods Digest, Vol 19,Issue 1-younes sadramehr SDS-PAGE repurification



Message: 2
Date: Thu, 30 Nov 2006 13:02:14 -0800 (PST)
From: younes sadramehr
Subject: SDS PAGE
To: [Only registered users see links. ]
Message-ID: <[Only registered users see links. ].re1.yahoo.com>
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Hi ..........
We had run a total protein SDS PAGE . After staining we saw a new & different band in one of the PAGE columns . Now we want to repurificate the protein ( the new band ) from the poly acrylamide gel . how should we do it ??we need the protein to re run on low voltage SDS PAGE for better isolation . how can we elute the protein in an economical & fast way ???/
thanks for your help .

Dear Younes,

here is a reference I found. One of the people in my lab does this often and it works well. I hope it helps.

Cheers,
Jess White
University of Texas- Austin
Dept of Medicinal Chemistry
Hook 'em Horns!


1: Electrophoresis. 1996 May;17(5):848-54.
Direct isolation of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analysis by electrospray-ionization mass spectrometry.

* Schuhmacher M,
* Glocker MO,
* Wunderlin M,
* Przybylski M.

Faculty of Chemistry, University of Konstanz, Germany.

A new method for the isolation of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using electroelution with a modified buffer system, is described. The method is suitable for direct characterization by electrospray-ionization mass spectrometry (ESI-MS), or alternatively matrix-assisted laser desorption-ionization (MALDI), no additional purification steps being required. Following separation by SDS-PAGE, proteins were stained with Coomassie Blue, and isolated by electroelution using SDS-free ammonium acetate (pH 2.5) as elution buffer. The polarity of the electroelution system was reversed, which, upon in situ dissociation of protein-SDS complexes, resulted in migration of free proteins to the cathode under the conditions employed. Recovery rates of 25-58% were determined for model proteins. Analyses by ESI-MS provided exact molecular weight determinations of isolated proteins and of protein mixtures not resolved by SDS-PAGE.
Essentially SDS-free molecular ions were obtained, except for bovine serum albumin with one SDS-adduct. Charge distribution of molecular ions were similar to those of the native proteins. The effects of beta-mercaptoethanol (beta-ME) and dithiothreitol (DTT) during electrophoresis were studied with hen egg white lysozyme, revealing the formation of mixed disulfide adducts between proteins and reducing agents. In a first bioanalytical application, hemofiltrate proteins from a patient with uremia were separated by SDS-PAGE. An ESI-MS analysis of the proteins isolated from the two most intensive gel bands enabled exact molecular weight determinations, and demonstrated that a gel band of ca. 17 kDa consisted of two different proteins.






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