Amplexultrared soluble fluorophore

Direct binding duplex ELISAs were used to measure ASPH, Humbug, Notch 1, Jagged 1, HES-1, and HIF-1α protein expression. Target protein immunoreactivity was detected with unique unlabeled monoclonal antibodies, horseradish peroxidase-conjugated secondary antibody, and Amplex UltraRed soluble fluorophore (Invitrogen, Carlsbad, CA, USA), as described [20 (link),60 (link)]. To adjust for variability in sample loading, the results were normalized to large acidic ribosomal protein (RPLPO) (Proteintech Group Inc, Chicago, IL, USA) [48 (link),61 (link)] which was biotinylated and detected with streptavidin-conjugated alkaline phosphatase and the 4-Methylumbelliferyl phosphate (4-MUP) substrate. Fluorescence intensities (Amplex Red: Ex 560 nm/Em 590 nm; 4-MUP: Ex360/Em450) were measured in a SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA). Antibody omission controls were included. The calculated target protein/RPLPO ratios were used for our statistical analysis.

Direct binding duplex ELISAs measured immunoreactivity with results
normalized to large acidic ribosomal protein (RPLPO) [33 (link)]. Immunoreactivity to target proteins was
detected with horseradish peroxidase-conjugated secondary antibody and Amplex
UltraRed soluble fluorophore (Invitrogen, Carlsbad, CA). RPLPO antibody
(Proteintech Group Inc, Chicago, IL) was biotinylated, and its immunoreactivity
was detected with streptavidin-conjugated alkaline phosphatase and the
4-Methylumbelliferyl phosphate (4-MUP) substrate. Fluorescence intensities
(Amplex Red: Ex 565 nm/Em 595 nm; 4-MUP: Ex360/Em450) was measured in a
SpectraMax M5 (Molecular Devices, Sunnyvale, CA). Antibody omission controls
were included. The calculated target protein/RPLPO ratios were used for
inter-group comparisons.