Sodium salicylate for fluorographical detection of immunoprecipitated proteins in agarose gels

Abstract

The Copenhagen Laboratory, UniversityofFilm detection of compounds labeled with the weak beta-emitters 3H and I4C is demonstrated for electroimmunoprecipitated proteins in agarose gels by means of the water-soluble fluorophor sodium salicylate. The degree ofenhancement offilm detection of tritium and carbon-14 with 0.7 M sodium salicylate paralleled fluorography with diphenyloxazole on polyacrylamide gels. The method was optimized and the sensitivity, expressed as radioactivity per cm immunoprecipitate, was 0.9 nCi/cm (2000 dpm/cm) in 24 h for 3H and 0.4 nCi/cm (830 dpm/cm) in 18 h for I4C. The procedure was found superior to the commercially available enhancement solutions 'EnHance' and 'Amplify'. Salicylate fluorography on Kodak X-Omat L and AR-5 films was about 30 and 60 times more sensitive than Ultrofilm 3H. With 14C-labeled anti-antibodies rocket electroimmunoprecipitation corresponding to about 50-100 pg protein could be visualized in 7 days. Conclusively, the method is rapid (soaking and drying require about 30 min), sensitive, reproducible, cheap, and advantageous if the immunoplates are to be stained for protein after fluorography.

Immunoelectrophoretic Analysis of Membrane Glycoproteins in Cultured Human Endothelial Cells

“…Radiolabelling o f rabbit antisera against human fibronectin and mouse immunoglobulins with 14C was performed by carbamylating the proteins with K14CNO (18). The procedure yielded specific activities of 167 and 368 dpm/ng protein for the antisera against fibronectin and mouse immunoglobulins, respectively.…”

An unusual transmissible agent ? MaTu

“…As reference marker proteins served the Sigma kit (product MW-SDS-200). For fluorography we were using salicylate [5].…”

Stage-specific polypeptides and villin expression during the intestinal epithelium substitution of the metamorphosing amphibian

“…Protein extracts were precipitated with acetone at -20" C, and the pellet was resuspended in O'Farrell lysis buffer [27]. Fluorography of dried gels was performed with Kodak X-AR films [19]. Isoelectric focussing (IEF) of the proteins was done according to the method of O'Farrell [27], essentially as described except that: (a) the ampholytes had a 5% final concentration, and the mixture was composed of one volume of pH 3-10 from Serva (Heidelberg, Federal Republic of Germany) and four volumes of pH 5-7 from Serva, Pharmacia (Uppsala, Sweden) and LKB (Bromma, Sweden) products; (b) the gels were 12 cm long; (c) a voltage of 650 V was applied for 15 h, then 1000 V for 1 h; (d) the second-dimension sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) was performed using 12.5% polyacrylamide slab gels.…”

“…A voltage of 200V was applied for 1 h, then 400 V for 3 h. The method of Morrissey [25] for silver staining gels was then used. Fluorography of dried gels was performed with Kodak X-AR films [19]. After exposition at -70" C, the film was developed when the counts per minute (cpm) loaded, multiplied by the number of days exposed, equalled 1 x lo6 cpm.…”