Column fractions were bound to protein-binding ELISA plates (at a dilution of 1:4). After overnight coupling and blocking (with 1% (w/v) BSA in PBS for 2 h at room temperature (RT)), the bound material were labelled with primary antibodies against proteins including CD9 (R&D systems) and CD81 (AbD serotec) or HSA (human serum albumin) (250 ng/ml) (R&D systems) was added for 2 h at RT on a plate shaker. After three washes, goat anti-mouse-biotinylated antibody (Perkin Elmer) diluted 1:2500 was added for 1.5 h. After three washes, Europium-conjugated streptavidin was added for 45 min. After a final six washes, a signal was obtained using time-resolved fluorometry, measured using a Wallac Victor-II multi-label plate reader (PerkinElmer) [13 ].
Goat anti mouse biotinylated antibody
Manufactured by PerkinElmer
The Goat anti-mouse-biotinylated antibody is a secondary antibody that binds to mouse primary antibodies. The antibody is labeled with biotin, which can be used to detect and amplify the signal from the primary antibody in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Wallac victor 2 multilabel plate reader, by PerkinElmer (2 mentions)
Europium conjugated streptavidin, by PerkinElmer (2 mentions)
Human serum albumin (hsa), by R&D Systems (1 mentions)
Nanodrop nd 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Spectramax i3, by Molecular Devices (1 mentions)
3 protocols using goat anti mouse biotinylated antibody
1
Protein-Binding ELISA Protocol
2
Immunostaining of Protein Fractions
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Immunostaining was performed as previously described (26 (link)). Briefly, fractions were bound to protein-binding microtitre ELISA plates (Greiner Bio-One, Stonehouse, UK) at a dilution of 1:4. After overnight coupling and blocking (with 1% (w/v) BSA in PBS for 2 h at room temperature (RT)), the bound material was labelled with
primary antibodies including CD9, ApoB, THP (Tamm–Horsfall protein) (at concentrations of 1 µg/ml) or HSA (human serum albumin) (at 250 ng/ml) (R&D Systems) all for 2 h at RT on a plate shaker. After 3 washes, goat antimouse-biotinylated antibody (Perkin Elmer) diluted 1:2,500 was added for 1.5 h. After 3 washes, Europium-conjugated streptavidin (Perkin Elmer) was added for 45 min. Finally after 6 washes, specific signal was measured by time-resolved fluorometry using a Wallac Victor-II multilabel plate reader (PerkinElmer Life). The method is referred to as an “ELISA-like” assay in the text.
primary antibodies including CD9, ApoB, THP (Tamm–Horsfall protein) (at concentrations of 1 µg/ml) or HSA (human serum albumin) (at 250 ng/ml) (R&D Systems) all for 2 h at RT on a plate shaker. After 3 washes, goat antimouse-biotinylated antibody (Perkin Elmer) diluted 1:2,500 was added for 1.5 h. After 3 washes, Europium-conjugated streptavidin (Perkin Elmer) was added for 45 min. Finally after 6 washes, specific signal was measured by time-resolved fluorometry using a Wallac Victor-II multilabel plate reader (PerkinElmer Life). The method is referred to as an “ELISA-like” assay in the text.
3
Quantitative Analysis of EV Surface Markers
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The protein concentrations in the CTX EV samples were measured using a NanoDrop ND-8000 spectrophotometer (Thermo Scientific), which ranged from approximately 250 to 600 µg mL -1 .
A microplate-based assay to detect the EV surface markers was employed. Briefly, the EV samples were bound to high protein binding microtitre ELISA plates (Greiner Bio-One) at a concentration of 1 mg mL -1 . After overnight incubation to allow the EVs to bind, blocking with 1% (w/v) BSA in PBS was carried out for 2 h at room temperature. The bound and blocked material was washed and consequently labelled with primary antibodies which are specific for the markers of interest at a concentration of 1 mg mL -1 for 2 h at room temperature on a plate shaker. After three washes, goat anti-mouse biotinylated antibody (PerkinElmer) was diluted at a ratio of 1:1000 and added to the plate for 1 h. After another three washes, Europium-conjugated streptavidin (PerkinElmer) diluted in a red assay buffer (Kaivogen) was added for 45 min. Finally, after six washes and the addition of a fluorescence intensifier solution (Kaivogen), the specific signal was measured using time-resolved fluorometry using an i3 Spectramax plate reader (Molecular Devices). 1,60
A microplate-based assay to detect the EV surface markers was employed. Briefly, the EV samples were bound to high protein binding microtitre ELISA plates (Greiner Bio-One) at a concentration of 1 mg mL -1 . After overnight incubation to allow the EVs to bind, blocking with 1% (w/v) BSA in PBS was carried out for 2 h at room temperature. The bound and blocked material was washed and consequently labelled with primary antibodies which are specific for the markers of interest at a concentration of 1 mg mL -1 for 2 h at room temperature on a plate shaker. After three washes, goat anti-mouse biotinylated antibody (PerkinElmer) was diluted at a ratio of 1:1000 and added to the plate for 1 h. After another three washes, Europium-conjugated streptavidin (PerkinElmer) diluted in a red assay buffer (Kaivogen) was added for 45 min. Finally, after six washes and the addition of a fluorescence intensifier solution (Kaivogen), the specific signal was measured using time-resolved fluorometry using an i3 Spectramax plate reader (Molecular Devices). 1,60
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