A simple enrichment procedure improves detection of membrane proteins by immunoblotting
| Autor: | Azamat V. Karginov, Michael O. Agaphonov |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Gel electrophoresis 030102 biochemistry & molecular biology biology Chemistry Immunoblotting Saccharomyces cerevisiae Membrane Proteins biology.organism_classification Pichia General Biochemistry Genetics and Molecular Biology Yeast Fungal Proteins 03 medical and health sciences Electrophoresis 030104 developmental biology Tubulin Membrane protein Biochemistry Immunoblot Analysis Protein purification biology.protein Electrophoresis Polyacrylamide Gel Biotechnology |
| Zdroj: | BioTechniques. 61:260-261 |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/000114474 |
| Popis: | We developed a novel approach to improve detection of membrane-associated proteins in yeast cell lysates by immunoblotting. Our method consists of a simple enrichment procedure using sedimentation to remove soluble proteins and the use of an alternative electrophoresis sample buffer, which allows for protein solubilization without heating. The efficacy of this approach was demonstrated for membrane proteins in Hansenula polymorpha (Pho87, Gas1, and Pmr1) and Saccharomyces cerevisiae (Gas1). Immunoblot analysis of proteins that are not membrane-associated showed that the precipitate fraction was depleted of Sup45, carboxypeptidase Y, and Hog1; however, tubulin and, to some extent, Sup35 and Tpd3 were precipitated together with the membrane proteins. The presence of tubulin in the same fraction as the membrane proteins allows its use as a reference protein. |
| Databáze: | OpenAIRE |
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