Using light scattering to locate less than a microgram of protein per band in polyacrylamide tube gels after isoelectric focusing.
After separation by isoelectric focusing (IEF) or non-equilibrium pH gradient electrophoresis (NEPHGE) in 9.2 M urea polyacrylamide tube gels, proteins with Mr > 20,000 can be precipitated by shaking the gels in 25% methanol. When a fiber-optic illuminator is placed in contact with the end of the gel, the cylindrical gel transmits light by internal reflection. In regions of the gel where protein is precipitated, this light is scattered, making it visible when the gel is viewed in a darkened room against a black background. The sensitivity of the method is moderate: less than 1 microgram protein per band (in a 3-mm diameter gel) can be detected. Because the protein in the bands precipitates rapidly (30 min), the solution used (25% methanol) is fairly benign to proteins, and the apparatus is not expensive, this technique should be useful in several situations including electrophoretic purification schemes. This method is especially useful for evaluating the quality of an IEF or NEPHGE tube gel before using it for the second dimension of a two-dimensional gel.
Driska
S P
SP
Physiology Department, Temple University Medical School, Philadelphia, Pennsylvania 19140, USA.
D000180:Acrylic Resins / Q000302:isolation & purification*; D000818:Animals; D001557:Benzenesulfonates; D002417:Cattle; D011232:Chemical Precipitation; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007525:Isoelectric Focusing / Q000379:methods*; D008027:Light; D000432:Methanol; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012459:Salicylates; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity; D012997:Solvents
D000180:Acrylic Resins; D001557:Benzenesulfonates; D010455:Peptides; D011506:Proteins; D012459:Salicylates; D012997:Solvents; C016680:polyacrylamide gels; C003366:sulfosalicylic acid; D000432:Methanol