Testing NF-kappa B1-p50 antibody specificity using knockout mice.
Cell extracts from knockout mice can provide definitive proof of antibody specificity. Two NF-kappa B1-p50 antibodies, sc-114 (a commercial antibody) and NR1157, were observed to recognize proteins having distinct electrophoretic mobilities of 52-55 kD and 50 kD, respectively, by Western blot analysis. In order to discriminate the specificity of these antibodies for NF-kappa B1-p50, whole cell extracts derived from NF-kappa B1-p105 knockout mice were employed. While the NR1157 antibody completely failed to recognize its 50 kD product in p105-/- knockout extracts, the sc-114 antibody still strongly recognized its 52-55 kD product. These data demonstrate that NR1157, but not sc-114, is highly specific for NF-kappa B1-p50 by Western blot analysis. In addition, these results highlight the utility of knockout cell extracts for discerning antibody specificity.
Pereira
D S
DS
Department of Biology, McMaster University, Hamilton, Ontario, Canada.
Kushner
D B
DB
Ricciardi
R P
RP
Graham
F L
FL
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D002457:Cell Extracts / Q000032:analysis; D004586:Electrophoresis; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000032:analysis / Q000276:immunology*; D052002:NF-kappa B p50 Subunit; D051381:Rats
D000906:Antibodies; D002457:Cell Extracts; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit