Rabbit anti gfp antibody

Smears of cultured PyXNL parasites were fixed on glass slides with ice-cold acetone for 5 min and blocked with PBS containing 5% nonfat milk at 37 °C for 30 min. They were then incubated with rabbit anti-CryPH antibodies (1: 1000), rabbit anti-GFP antibody (1: 500, Clontech, Mountain View, CA, USA) and mouse anti-Pys25 mAb (1: 20,000) at 37 °C for 1 h, and thereafter with Alexa Fluor 488-goat anti-rabbit IgG antibody and Alexa Fluor 546-goat anti-mouse IgG antibody (Invitrogen) as secondary antibodies (1:500 dilution) at 37 °C for 30 min, together with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI). After mounting in ProLong Gold antifade reagent (Invitrogen), samples were observed with an inverted fluorescence microscope (Axio observer z1, Carl Zeiss, Oberkochen, Germany), and images were taken using AxioVision software (Carl Zeiss).

Mouse monoclonal anti-Rac1 antibody was purchased from Cytoskeleton Inc. (Denver, CO, USA). Rabbit anti-GFP antibody was from Clontech (Mountain View, CA, USA). Mouse monoclonal anti-pRac1S71, anti-14-3-3s, 14-3-3η, -γ, -σ, and -θ, and anti-GST antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit monoclonal anti-α-tubulin antibody was from Abcam (Abcam Inc, Toronto, ON, Canada). FITC- and TRITC-conjugated donkey anti-mouse and anti-rabbit antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Glutathione cross-linked to 4% agarose, goat anti-mouse IgG conjugated with agarose, and protein A conjugated with agarose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mammalian Protein Extraction Reagent (M-Per) was purchased from Thermo Fisher Scientific Inc. (Rockford, IL USA). Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich.

For preparation of whole cell lysates, plates were washed three times with ice-cold phosphate-buffered saline (pH 7.2), drained on ice and then scraped off in lysis buffer (140 mmol/L NaCl, 3 mmol/L MgCl₂, 1 mmol/L dithiothreitiol and 0.5% Nonidet-P40 in a 20 mmol/L sodium phosphate buffer, pH 8.0) or, for IP experiments, in IP lysis buffer (50 mmol/L NaCl, 10% glycerol, 1% Nonidet-P40 in a 10 mmol/L sodium phosphate buffer, pH 8.0). Lysis buffers were supplemented with protease inhibitors. Cell lysis was performed on ice for 30 minutes. Lysates were subsequently cleared by centrifugation (16,000 rcf for 10 minutes at 4 °C). For IP experiments, lysates in IP lysis buffer were incubated rotating at 4 °C over night in the presence of anti-acetylated lysine antibody (5 μl antibody for 500 mg total protein) crosslinked to Protein G-dynabeads (R) (Invitrogen). For control IPs, rabbit anti-GFP antibody (Clontech, Mountain View, CA) crosslinked to protein G-dynabeads (R) was used. The immobilized antigen-antibody complexes were washed three times with IP lysis buffer. Bound proteins were eluted from the immobilized antibodies with sodium dodecylsulfate (SDS) sample buffer and further processed for western blotting.

The brains of male and female silkmoths were dissected and fixed in 4% paraformaldehyde/phosphate-buffered saline overnight at 4°C. The brains were washed three times in phosphate-buffered saline containing 0.3% TritonX-100 (PBTX), blocked in 7% normal donkey serum in PBTX for 3 h at room temperature, and incubated in rabbit anti-GFP antibody (1/200; Clontech, Mountain View, CA), anti-synapsin monoclonal antibody (1/100; Developmental Studies Hybridoma Bank, Iowa City, IA), and 1% normal donkey serum for 1 week at 4°C. After several washes in PBTX, signals were developed by incubation with fluorescein isothiocyanate-conjugated anti-rabbit IgG (1/200; Cappel, Aurora, OH) and TexasRed-conjugated anti-mouse IgG (1/200; Cappel) for two overnights at 4°C. The nuclear DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) and pictures were obtained using the confocal microscope LSM5 (Carl Zeiss, Germany).