TY - JOUR
T1 - Detection of low-molecular-weight polypeptides on nitrocellulose with monoclonal antibodies
AU - Rosenbaum, Lawrence C.
AU - Nilaver, Gajanan
AU - Hagman, Heidi M.
AU - Neuwelt, Edward A.
N1 - Funding Information:
We thank Gary L. Peterson and Dean A. Malencik for their expert review and comments on this manuscript. This work was supported by PHS Grant DK-37205, Veterans Administration Merit Review Grant, the National Institutes of Health Grant 5ROl CA31770-07, and Oncogen of Seattle, Washington. H.M.H. was the recipient of N. L. Tartar Research Fellowship.
PY - 1989/12
Y1 - 1989/12
N2 - 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 β-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.
AB - 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 β-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.
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U2 - 10.1016/0003-2697(89)90475-2
DO - 10.1016/0003-2697(89)90475-2
M3 - Article
C2 - 2696385
AN - SCOPUS:0024832007
SN - 0003-2697
VL - 183
SP - 250
EP - 257
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -