Measurement of specific radioactivity in proteins separated by two-dimensional gel electrophoresis.
We report a method to quantify the specific radioactivity of proteins that have been separated by 2-DE. Gels are stained with SyproRuby, and protein spots are excised. The SyproRuby dye is extracted from each spot using DMSO, and the fluorescence is quantified automatically using a plate reader. The extracted gel piece is then dissolved in hydrogen peroxide and radioactivity is quantified by liquid scintillation counting. Gentle agitation with DMSO for 24 h was found to extract all the SyproRuby dye from gel fragments. The fluorescence of the extract was linearly related to the amount of BSA loaded onto a series of 1-D gels. When rat muscle samples were run on 2-DE gels, the fluorescence extracted from 54 protein spots showed a good correlation (r = 0.79, p < 0.001) with the corresponding spot intensity measured by conventional scanning and image analysis. DMSO extraction was found not to affect the amount of radioactive protein left in the gel. When a series of BSA solutions of known specific radioactivity were run on 2-DE gels, the specific radioactivity measured by the new method showed a good correlation (r = 0.98, p < 0.01, n = 5) with the specific radioactivity measured directly before loading. Reproducibility of the method was measured in a series of 2-DE gels containing proteins from the livers of rats and mice that had been injected with [35S]methionine. Variability tended to increase when the amount of radioactivity in the protein spot was low, but for samples containing at least 10 dpm above background the CV was around 30%, which is comparable to that obtained when measuring protein expression by conventional image analysis of SyproRuby-stained 2-DE gels. Similar results were obtained whether spots were excised manually or using a spot excision robot. This method offers a high-throughput, cost-effective and reliable method of quantifying the specific radioactivity of proteins from metabolic labelling experiments carried out in vivo, so long as sufficient quantities of radioactive tracer are used.
Nutritional Sciences Research Division, King's College London, London, UK.
Shaobo
S
Zhou
Christopher J
CJ
Mann
Michael J
MJ
Dunn
Victor R
VR
Preedy
Peter W
PW
Emery
D000818:Animals; D002417:Cattle; D004121:Dimethyl Sulfoxide; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005260:Female; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D008099:Liver / Q000737:chemistry; D051379:Mice; D009124:Muscle Proteins / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D011868:Radioisotopes / Q000032:analysis*; D051381:Rats; D015203:Reproducibility of Results; D012371:Robotics; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D013462:Sulfur Radioisotopes / Q000032:analysis
D005456:Fluorescent Dyes; D009124:Muscle Proteins; D011506:Proteins; D011868:Radioisotopes; D013462:Sulfur Radioisotopes; D012710:Serum Albumin, Bovine; D004121:Dimethyl Sulfoxide