Visualization of alkaline phosphatase-labelled antibodies on immunoblots by means of formazan staining using indoxyl phosphate and thiazolyl blue.
Alkaline phosphatase-conjugated secondary antibodies on blotting membranes were visualized by means of a modified formazan staining method (Hodson & Skillen 1988). It is based on the standard 5-bromo-4-chloro-3-indolyl phosphate/Nitroblue tetrazolium method, but employs different buffer conditions and thiazolyl blue instead of Nitroblue. It gave a sensitivity of 1 ng albumin in a 12 mm2 dot and less background staining on dot blots than the standard method. The new protocol provides an advantage with regard to ease of staining and preparation of reagents while it equals the former method with respect to sensitivity.
Heegaard
N H
NH
Blood Bank, Rigshospitalet, Copenhagen, Denmark.
D000469:Alkaline Phosphatase; D000906:Antibodies; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000648:ultrastructure; D005562:Formazans; D015151:Immunoblotting; D007211:Indoles; D011143:Polyvinyl Chloride / Q000031:analogs & derivatives; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000737:chemistry; D013194:Staining and Labeling; D013778:Tetrazolium Salts; D013844:Thiazoles
D000906:Antibodies; D005562:Formazans; D007211:Indoles; D012709:Serum Albumin; D013778:Tetrazolium Salts; D013844:Thiazoles; C034082:indoxyl; C029547:polyvinylidene chloride; D011143:Polyvinyl Chloride; D003101:Collodion; D000469:Alkaline Phosphatase; C022616:thiazolyl blue