GFP ELISA
GFP ELISA – the “home-made” protocol
Reagents
96-well plates Immulon 2HB
Goat anti-GFP polyclonal IgG (Rockland), 1 mg/ml, aliquot & store -20oC.
Rabbit anti-GFP polyclonal IgG (Abcam, Ab290). Store as 1:160 dilution in 50 % glycerol at -20oC.
Goat anti-rabbit horseradish peroxidase (Biorad), 4oC.
Purified GFP protein at 1 mg/ml (Clontech). Dilute to 10 ng/µl (1:100) & 100 pg/µl (1:10,000) in Blocking buffer for standards. Store -70oC.
Substrate ABTS (Sigma). 100 ml = 100 mg ABTS + 1.67 g Buffer powder (Roche).
Blocking buffer: 10 mg/ml Blocking Reagent (ROCHE) in PBS. Store at -20oC.
PBS-Tween from media prep.
Coating buffer: 0.34 % w/v Na2CO3 (anhydrous), 0.57 % NaHCO3, pH 9.6.
Preparing plates
Coat required number of wells with 0.1 ml/well Goat anti-GFP:
Dilute IgG to 1:3000 in Coating Buffer and apply to plate.
Bind 4oC overnight.
Wash 1X PBS-T.
Block with 0.2 ml/well Blocking buffer, 2 hr room temp.
Shake off buffer and blot dry.
Wrap in cling-film and store at -20oC.
[Plates keep for >2 weeks, possibly indefinitely].
GFP ELISA
Re-hydrate coated wells with 0.2 ml/well PBS-T.
Dilute GFP standards (from 0 to 1000 pg) and samples in Blocking buffer to 200 µl and add to wells.
Incubate 1 hr 37oC.
Wash 5x with 200-250 µl PBS-Tween.
Add Ab290 polyclonal, fresh 1:100 dilution in Blocking buffer, 200 µl / well (final concentration 1:16,000).
Incubate 1 hr 37oC.
Wash 5x with 200-250 µl PBS-Tween.
Add GAR-POD, fresh 1:1000 dilution in Blocking buffer, 200 µl / well.
Incubate 1 hr 37oC.
Wash 5x with 200-250 µl PBS-Tween.
Add ABTS substrate 200 µl / well, incubate at room temp until standards sufficiently green.
Read OD405 in ELISA plate reader.