Proteome resolution by two-dimensional gel electrophoresis varies with the commercial source of IPG strips.
By facilitating reproducible first dimension separations, commercial immobilized pH gradient (IPG) strips enable high throughput and high-resolution proteomic analyses using two-dimensional gel electrophoresis (2DE). Amersham, Biorad, Invitrogen, and Sigma all market linear pH 3-10 IPG strips. We have applied optimized 2DE protocols with both membrane and soluble brain protein extracts to critically evaluate all four products. Resolved protein spots were quantitatively evaluated after carrying out these protocols using IPG strips from the four companies. Biorad and Amersham IPG strips resolved a high number of membrane and soluble proteins, respectively. Furthermore, Amersham IPG strips eluted the largest amount of protein into the second dimension gels and had the most protein remaining in the strip after 2DE. Biorad and Amersham IPG strips maintained a consistent linear pH 3-10 gradient, whereas those from Invitrogen appeared nonlinear or "compressed" within the central pH region. The gradient range within Sigma IPG strips appeared to be slightly less than pH 3-10, due to one extended pH unit within the gradient. Overall, all four commercially available IPG strips have the ability to resolve both membrane and soluble brain proteomes. The difference is that Amersham and Biorad do so more consistently and with better spot resolution. It appears that the physical/chemical nature of commercially available IPG strips can vary considerably, leading to marked differences in subsequent protein resolution in 2DE. These differences likely reflect variations in the uptake of proteins into the strips, and differences in the focusing and elution of proteins from the first to the second dimension. These differences would appear, in part, to underlie some inter-lab variations in the effective resolution of proteomes.
Taylor
Regan C
RC
Hotchkiss Brain Institute, and Department of Physiology & Biophysics, University of Calgary Faculty of Medicine, 3330 Hospital Drive NW, Calgary AB, T2N 4N1, Canada.
Coorssen
Jens R
JR
D000818:Animals; D001923:Brain Chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D011934:Reagent Strips
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D011934:Reagent Strips