Detection of low-molecular-weight polypeptides on nitrocellulose with monoclonal antibodies.
An immunoblotting method to detect low-molecular-weight peptides with monoclonal antibodies that normally fail to demonstrate immunoreactivity using conventional blotting techniques is described. Detection of neurophysin, insulin, calcitonin, vasopressin, and beta-endorphin electroblotted on nitrocellulose membranes was optimized after introducing four modifications into the conventional procedure. These include renaturing the gels after sodium dodecyl sulfate electrophoresis, electroblotting the renatured gels in basic transfer buffer, fixing and/or heating the blots, and using avidin/alkaline phosphatase conjugates for antigen/antibody detection. This technique likely enables the denatured peptides to regain their native conformation and, therefore, restores antigenicity and recognition by highly structural specific monoclonal antibodies. Although the most dramatic improvement with this technique is with monoclonal antibodies, a modest improvement in sensitivity can be obtained when immunoblots are probed with polyclonal antibodies. The high resolution of this system will be useful in probing blots of partial proteolytic digests of proteins with both monoclonal and polyclonal antibodies.
Rosenbaum
L C
LC
Oregon Health Sciences University, Department of Biochemistry, Portland 97201.
Nilaver
G
G
Hagman
H M
HM
Neuwelt
E A
EA
D000469:Alkaline Phosphatase; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal; D001360:Avidin; D002021:Buffers; D002116:Calcitonin / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting / Q000379:methods; D007328:Insulin / Q000032:analysis; D008970:Molecular Weight; D009481:Neurophysins / Q000032:analysis; D010455:Peptides / Q000032:analysis* / Q000276:immunology; D013552:Swine; D014667:Vasopressins / Q000032:analysis
D000906:Antibodies; D000911:Antibodies, Monoclonal; D002021:Buffers; D007328:Insulin; D009481:Neurophysins; D010455:Peptides; D014667:Vasopressins; D001360:Avidin; D002116:Calcitonin; D000469:Alkaline Phosphatase