GFP ELISA – the “home-made” protocol

Reagents

96-well plates Immulon 2HB

Goat anti-GFP polyclonal IgG (Rockland), 1 mg/ml, aliquot & store -20^oC.

Rabbit anti-GFP polyclonal IgG (Abcam, Ab290). Store as 1:160 dilution in 50 % glycerol at -20^oC.

Goat anti-rabbit horseradish peroxidase (Biorad), 4^oC.

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 -70^oC.

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 -20^oC.

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 4^oC 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 -20^oC.

[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 37^oC.

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 37^oC.

Wash 5x with 200-250 µl PBS-Tween.

Add GAR-POD, fresh 1:1000 dilution in Blocking buffer, 200 µl / well.

Incubate 1 hr 37^oC.

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.