The staining of acidic proteins on polyacrylamide gels: enhanced sensitivity and stability of "Stains-all" staining in combination with silver nitrate.
A number of acidic proteins, such as those found in bone and dentin, are poorly resolved on acrylamide gels using Coomassie blue or silver nitrate staining. The cationic dye Stains-all allows visualization and identification of these proteins due to their differential staining: highly acidic proteins stain blue and intact proteoglycans stain purple, whereas less acidic proteins stain pink. However, the use of Stains-all is limited due to relatively poor staining sensitivity and lack of stability to light. A procedure which addresses these deficiencies has been developed utilizing established protocols for Stains-all staining followed by silver nitrate incubation and development. In this way, phosphoproteins such as osteopontin, bone sialoprotein, dentin phosphophoryn, and other acidic glycoproteins are visualized at higher sensitivity (greater than fivefold) and staining stability than normally achieved with just Stains-all. The protocol stains a greater variety of proteins than a combined alcian blue/silver staining procedure previously described. Utilizing the Stains-all/silver protocol, porcine bone osteopontin, a protein not visualized by standard silver staining, can be observed in amounts as little as 0.25 ng on polyacrylamide gels. Furthermore, densitometric scans demonstrate that the staining intensity is proportional to osteopontin amount and can be used for quantification over a range from 0.25 to 50 ng.
Goldberg
H A
HA
Skeletal Biology Group, The University of Western Ontario, London, Ontario, N6A 5C1, Canada. hagoldbe@julian.uwo.ca
Warner
K J
KJ
D000180:Acrylic Resins; D000818:Animals; D002232:Carbocyanines; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis; D058574:Integrin-Binding Sialoprotein; D053495:Osteopontin; D010750:Phosphoproteins / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D012795:Sialoglycoproteins / Q000032:analysis; D016622:Silver Staining / Q000379:methods*; D013552:Swine
D000180:Acrylic Resins; D002232:Carbocyanines; D004396:Coloring Agents; D006023:Glycoproteins; C546871:IBSP protein, human; D058574:Integrin-Binding Sialoprotein; D010750:Phosphoproteins; D011506:Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C017630:phosphophoryn; C016680:polyacrylamide gels; D053495:Osteopontin; C012040:stains-all