Reducing the artifacts produced by impure antisera in immunoblots of low-molecular-mass proteins in urine
Perini, J.M.; Dehon, B.; Marianne, T.; Klein, A.; Roussel, P.
Clinical Chemistry 32(5): 811-815
1986
ISSN/ISBN: 0009-9147 PMID: 2421944 Document Number: 267761
Immunoblots of several urinary low-molecular-mass proteins can be very useful in investigations of pathological proteinuria. However, use of certain commercial antisera in such procedures leads to artifacts corresponding to nonspecific bands; e.g., immunoglobulins from nonimmunized rabbit serum may bind to human urinary proteins, and this binding is not inhibited by Triton X-100. We have developed a procedure to improve the specificity of detection of urinary low-Mr proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by immunoblotting with commercial antisera: we treat the protein blot with a mixture of mercaptoethanol and sodium dodecyl sulfate before incubation with the first antiserum. This allows direct use of commercial antisera without prior absorption of contaminating antibodies.