Sodium dodecyl sulfate enhancement of quantitative immunoenzyme dot-blot assays on nitrocellulose.
Treating proteins with low concentrations of sodium dodecyl sulfate (SDS) and boiling for 2-3 min increased the linear range and total amount of protein that could be bound to nitrocellulose. Human serum albumin (HSA) and cathepsin G (Cat G) were both optimally bound at an SDS concentration of 10 micrograms/ml, while bronchial leukocyte proteinase inhibitor (BLPI) required 50 micrograms/ml SDS for optimum binding, corresponding to SDS-to-protein weight ratios of 0.5 and 2.5, respectively. Ionic strength and pH of the blotting buffers had a greater effect on the binding of SDS-treated proteins than on native proteins, with the linear binding range and total capacity for SDS-treated proteins being increased. Boiling SDS-treated human leukocyte extracts inactivated endogenous peroxidases, eliminating their interference with peroxidase-linked secondary antibodies in immunoassays. The nonionic detergents, Tween 20 and Nonidet P-40, were shown to rapidly wash both native and SDS-treated HSA off the filters, but these HSA samples were stable to washing with SDS. Although SDS-treated Cat G was more stable with nonionic detergents than was native Cat G, it was less resistant to washing with SDS. The substitution of SDS for nonionic detergents improved the response of immunoassays with native and SDS-treated proteins. Affinity-purified antibodies to human mast cell tryptase cross-reacted with native Cat G, but not with SDS-treated Cat G, indicating that SDS treatment can improve the specificity of immunoassays employing polyclonal antisera. These effects appear to be the result of partial denaturation and increases in the hydrophobicity of SDS-treated relative to native proteins.
Smith
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Department of Biochemistry, Quillen-Dishner College of Medicine, East Tennessee State University, Johnson City 37614.
Musich
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Johnson
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D002021:Buffers; D003101:Collodion; D003429:Cross Reactions; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting / Q000379:methods*; D007124:Immunoenzyme Techniques; D011506:Proteins / Q000032:analysis*; D012965:Sodium Chloride; D012967:Sodium Dodecyl Sulfate
D002021:Buffers; D003902:Detergents; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D012965:Sodium Chloride; D003101:Collodion