A novel approach to enhance antibody sensitivity and specificity by peptide cross-linking.
Most current techniques employed to improve antigen-antibody signals in Western blotting and in immunohistochemistry rely on sample processing prior to staining (e.g., microwaving) or using a more robust reporter (e.g., a secondary antibody with biotin-streptavidin). We have developed and optimized a new approach intended to stabilize the complexes formed between antigens and their respective primary antibodies by cupric ions at high pH. This technique improves the affinity and lowers cross-reactivity with nonspecific bands of approximately 20% of antibodies tested (5/25). Here we report that this method can enhance antigen-antibody specificity and can improve the utility of some poorly reactive primary antibodies.
Namiki
Takeshi
T
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Building 37, Room 3132, Bethesda, MD 20814, USA.
Valencia
Julio C
JC
Hall
Matthew D
MD
Hearing
Vincent J
VJ
D000818:Animals; D000915:Antibody Affinity / Q000187:drug effects; D000918:Antibody Specificity / Q000187:drug effects; D000937:Antigen-Antibody Reactions / Q000187:drug effects; D001738:Biuret Reaction; D003300:Copper / Q000737:chemistry* / Q000494:pharmacology; D003429:Cross Reactions / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D051379:Mice; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*
D003432:Cross-Linking Reagents; D010455:Peptides; D003300:Copper
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