Rabbit anti gfp

To extract total leaf protein, 100 mg of agroinfiltrated leaf tissue was ground to a powder with a pestle and liquid nitrogen. The powdered sample was homogenized in protein extraction buffer [10% glycerol, 25 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM ethylenediaminotetraacetic acid (EDTA), 0.15% IGEPAL ® CA-630 (Octylphenoxy poly(ethyleneoxy)ethanol, branched), 10 mM dithiothreitol, and protease inhibitor cocktail (Roche)] and centrifuged for 4 min at 12,000 g at 4°C, to pellet the crude extract and the supernatant collected.
Protein extracts were analyzed on an SDS-PAGE gel (46) (link) and electrophoretically transferred onto a nitrocellulose membrane (47) (link). For immunological detection of GFP-tagged and RFPtagged proteins, rabbit anti-GFP (1:1000) and mouse anti-RFP (1:2000) (ChromoTek) monoclonal primary antibodies were used in combination with anti-rabbit or anti-mouse IgG horseradish peroxidase conjugated secondary antibodies. The blotted membrane was incubated with Pierce Enhanced Chemiluminescence ECL-Plus substrate and chemiluminescence signals imaged using X-ray film (Fujifirm, Toyo, Japan).