Fig. 1
Antibody-independent signals in blots developed using Neutravidin-HRP. a Left, Western blot of mouse-brain extracts, probed with biotin-conjugated 6E10 (6E10-biotin), followed by Neutravidin-HRP (NA-HRP) and chemiluminescence reagent. Right, samples immunoprecipitated with 6E10 prior to blotting. Note bands marked by asterisks (*), which are eliminated (~ 46 kDa, ~ 55 kDa) or diminished (~ 75 kDa, ~ 150 kDa) when samples are immunoprecipitated prior to blotting. **, Protein G. b Blot of mouse-brain extracts processed with NA-HRP and chemiluminescence reagent, without exposure to antibody 6E10. Note bands marked by (*), which appear even in the absence of antibody. +, APP transgenic mice (line Tg6209); −, non-transgenic mouse. c Western blot of extracts from Alzheimer’s disease (AD) brains, probed with 6E10-biotin, followed by NA-HRP and chemiluminescence reagent. Lanes 1 and 2, brains had AD and Lewy body (LBD) pathology; lane 3, brain displayed AD pathology and atherosclerosis (ATS). Bands marked by asterisks (*) are also present in (e). (s)APP, full-length and/or α-secretase-cleaved, soluble N-terminal fragment of APP; Aβ, monomeric Aβ. d Brain extracts in (c) immunoprecipitated with 6E10; blot processed as in (c). Compare pattern of bands in (c) and (d). (s)APP, Aβ, and a doublet at ~ 12 kDa are immunoprecipitated. **, Protein G. e Blot processed with NA-HRP and chemiluminescence reagent, without exposure to antibody 6E10. IP, sample immunoprecipitated with 6E10; other lanes, samples prepared as in (c). Note that (s)APP, ~ 12-kDa doublet, and Aβ bands, seen when blots are exposed to anti-APP/Aβ antibody 6E10 (c), are absent here, and that immunoprecipitation eliminates or diminishes the non-specific bands (*). f Left, blot probed with unconjugated 6E10 followed by goat-anti-mouse IgG conjugated to HRP (GαM IgG-HRP), 1-h exposure. Only the (s)APP band is detectable. Right, (s)APP signal after 1-h exp. in blot probed using 6E10-biotin, followed by NA-HRP, for comparison. (Bottom panels accompanying Western blots show anti-α-tubulin loading controls (see Methods))