An electrophoretic approach to screen for glutamine deamidation.
Protein deamidation is a posttranslational modification with important implications in physiology and medicine. There is, however, no simple technique for a rapid screening of protein deamidation. The deamidating activity of transglutaminase was applied to establish a simple method for the screen of protein deamidation using recombinant human growth hormone, a rat hippocampal membrane fraction, and a cell homogenate enriched in 5-hydroxytryptamine-1A receptor as model systems. Here we report a simple, economic, and fast approach to assess protein deamidation by two electrophoretic methods: differential cleavage on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) via in situ V8 protease digestion and the principle of spot shifting via blue native (BN)-PAGE/two-dimensional (2D)-SDS-PAGE/immunoblotting.
Department of Pediatrics, Medical University of Vienna, 1090 Vienna, Austria.
Narkhyun
N
Bae
Jae-Won
JW
Yang
Harald
H
Sitte
Arnold
A
Pollak
Javier
J
Marquez
Gert
G
Lubec
D000577:Amides / Q000378:metabolism*; D000818:Animals; D002021:Buffers; D002457:Cell Extracts; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005973:Glutamine / Q000032:analysis*; D019382:Human Growth Hormone / Q000378:metabolism; D006801:Humans; D008566:Membranes / Q000378:metabolism; D051381:Rats; D044282:Receptor, Serotonin, 5-HT1A / Q000378:metabolism; D013194:Staining and Labeling; D013347:Subcellular Fractions / Q000378:metabolism
D000577:Amides; D002021:Buffers; D002457:Cell Extracts; D005973:Glutamine; D044282:Receptor, Serotonin, 5-HT1A; D019382:Human Growth Hormone