Direct dye binding--a quantitative assay for solid-phase immobilized protein.
A direct dye-binding procedure was established for the quantification of protein after its immobilization on a solid phase, using IgG and BSA as model proteins. The assay, which in the range 0-5 mg protein/ml gel correlates well with indirect protein determination by A280 as well as determination of protein hydrolyzed from the gel, is based on a modified Bradford dye-binding assay. As the protein coupled to the gel binds the dye, a decrease in A465 of the supernatant is measured. Three solid supports commonly used for protein immobilization (Sepharose, Sephadex, Sephacryl) were found to be compatible with the dye-binding assay while nonspecific dye binding was found to HEMA gels. Protein was coupled to Sephacryl S-1000 using three different activation methods (aldehyde, hydrazine, and adipic acid dihydrazide). Artifactual dye-binding was not observed using any of the three different "linkers." The assay is easily carried out and represents a useful tool, e.g., when optimizing procedures for protein immobilization.
Bonde
M
M
Department of Biochemistry and Nutrition, Technical University of Denmark, Lyngby.
Pontoppidan
H
H
Pepper
D S
DS
D004396:Coloring Agents / Q000378:metabolism*; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013057:Spectrum Analysis
D004396:Coloring Agents; D007074:Immunoglobulin G; D011506:Proteins; D012710:Serum Albumin, Bovine