Combined file of dot blot images and quantification

IGF peptides (recombinant mouse IGF-II or synthetic mouse vesiculin) were biotinylated following manufacturers’ instructions. Either 40ng or 20ng mouse IGFBP of each IGFBP (1, 2, 3, 5 and 6) was dotted (2µl) onto a nitrocellulose membrane and dried at 37oC for 5 minutes. The blots were incubated at room temperature with agitation, first in TBS with 3% BSA for 2 hours, then in TBS with 0.1% Tween 20 for 10 minutes. The blot was then incubated overnight with 40ng of biotinylated peptides in TBS with 1% BSA and 0.1% tween at 4oC with agitation. The blots were then washed three times with TBS with 1% BSA and 0.1% tween 20 for 5 minutes at room temperature before being incubated for 1 hour with Streptavidin-HPR (1:2000 dilution) in TBS with 1% BSA and 0.1% tween 20. A further wash preceded incubation with ECL reagent and imaging and densitometry analysis (Image lab, BioRad). For testing equal signal from both biotinylated peptides, different concentrations of both labelled peptides were directly dotted on the membrane which was dried, washed, incubated with Streptavidin-HPR, washed then detected.