Mouse monoclonal anti gfp antibody

1.10⁶ conidia were inoculated in liquid AMM and incubated on a rotary shaker at 220 rpm and 37°C for 16 h. Cells were harvested (0 min), transferred to fresh liquid AMM followed by induction with 100 μM BCS and 100 μM CuSO₄ for the period indicated in the figures. Mycelia was collected, lyophilized for 16 h and ground in a minibeater. Protein samples were extracted by alkaline-lysis buffer (0.2 M NaOH, 0.2% β-mercaptoethanol), as described in Hervas-Aguilar and Penalva (2010) (link).
Five micro liters of each protein sample were loaded into each well of 10% SDS-polyacrylamide gels and electrotransfered to Immun-Blot^® PVDF membranes by TransBlot Turbo Transfer System (Bio-Rad). Ponceau S staining was used as a loading control (0.1% Ponceau S, 5% acetic acid). Ponceau staining was removed using a 20% acetonitrile, 200 μM NaOH solution. For Western blotting the following primary antibodies were used: monoclonal mouse anti-GFP antibody (1/5000; Roche). As secondary antibodies peroxidase-conjugated goat anti-mouse IgG immunoglobulin (1/4000 dilution; Jackson Immunoresearch Lab.) is used. Proteins were detected using Clarity^TM Western ECL Substrate (Bio-Rad) in a Chemidoc + XRS system (Bio-Rad). Signal intensity was measured with Image Lab 3.0 software (Bio-Rad).

daf-2(e1370) animals expressing SPT-5::GFP were synchronized by bleaching, grown on HT115 bacteria without dsRNA-expressing plasmids at 15 °C until the L4 stage, washed in antibiotics to remove the HT115, and transferred to plates seeded with either control or spt-5 RNAi bacteria. 5-fluoro-2′-deoxyuridine (FUDR) was added to a final concentration of 50 µM to prevent progeny production and the plates were shifted to 25 °C. On day 2 of adulthood, 50 worms per condition were harvested and analyzed by SDS-PAGE and western blotting. For detection of SPT-5::GFP we used monoclonal mouse anti-GFP antibody (Roche) and for detection of actin we used monoclonal (C4) mouse anti-actin antibody—both diluted 1:1000 in TBST containing 5% milk powder. For quantification, band intensities were determined using the Fiji image processing package (ImageJ) and data were normalized to actin. This experiment was repeated 3 times. Unpaired t test (two-tailed) was used for comparisons and statistical analysis.

10⁷ cells were collected for each sample and total protein was extracted by TCA precipitation as described (Keaton et al., 2008 (link)). Electrophoresis and Western blotting were performed as described (Bose et al., 2001 (link)). Monoclonal mouse anti-Cdc42 antibodies (Wu and Brennwald, 2010 (link)) were used at 1:500 dilution. Monoclonal mouse anti-GFP antibody (Roche Applied Science, Germany) was used at a 1:1000 dilution. Polyclonal rabbit anti-Cdc11 antibody (Santa Cruz Biotechnology, Dallas, TX) was used at a 1:5000 dilution. Fluorophore-conjugated secondary antibodies against mouse (IRDye 800CW goat anti-mouse IgG, LI-COR Biosciences, Lincoln, NE) or rabbit (Alexa Fluor 680 goat anti-rabbit IgG, Invitrogen, Carlsbad, CA) antibodies were used at 1:5000 dilutions. Blots were visualized and quantified using the ODYSSEY imaging system (LI-COR Biosciences).

The following antibodies were used against Drosophila proteins: goat anti-biotin-horseradish peroxidase (HRP) conjugated antibody (Cell Signalling); chicken polyclonal anti-BirA antibody (Sigma); rabbit polyclonal anti-Parkin antibody [44 (link)]; mouse monoclonal anti-Syx1A antibody (DSHB); rabbit polyclonal anti-RdhB [45 (link)]; rabbit polyclonal anti-ArgK [46 (link)]; rabbit polyclonal anti-Vps4 [47 (link)]; rabbit polyclonal anti-Fax antibody (a gift from Eric Liebl); rabbit polyclonal anti-Ubiquitin antibody (Sigma). The following antibodies were used against Human proteins: goat polyclonal anti-VPS35 antibody (Abcam); mouse monoclonal anti-Cleaved Parp-85 fragment (Cell Signaling); mouse monoclonal anti-Parkin (Santa Cruz); rabbit polyclonal anti-PINK1 (Novus Biologicals); rabbit polyclonal anti-Miro1 (Sigma); rabbit polyclonal anti-Tim44 (Sigma); rabbit polyclonal anti-Tom20 (Sigma); mouse monoclonal (Abcam) and rabbit polyclonal (Sigma) anti-Actin. For monitoring the GFP pull-downs the following antibodies were used: monoclonal mouse anti-GFP antibody (Roche) and monoclonal mouse anti-Flag M2-HRP conjugated antibody (Sigma). Anti-mouse, rabbit and chicken HRP labelled secondary antibodies (Jackson ImmunoResearch Laboratories) and anti-guinea pig (Invitrogen) were used; and anti-mouse, rabbit and sheep IR 680 and IR800-coupled antibodies (LI-COR Biosciences).

Yeast cells were grown to log phase in SD media at 30 °C and 10 OD600/ml equivalents of cells were harvested and stored at –80 °C. Cells were thawed and lysed by vortexing in 100 μl of Thorner buffer (8 M Urea, 5% SDS, 40 mM Tris-Cl (pH 6.4), 1% beta-mercaptoethanol and 0.4 mg/ml bromophenol blue) with ~100 μl of acid-washed glass beads/sample at 70 °C for 5 min. Lysates were centrifuged at 14,000 RPM for 30 s and separated on 8% SDS-PAGE gels followed by western blotting with monoclonal mouse anti-GFP antibodies (11–814–460-001; Roche) or monoclonal mouse anti-PGK1 antibodies (AB_2532235, 22C5D8, Invitrogen), and secondary polyclonal goat anti-mouse antibodies conjugated to horseradish peroxidase (115–035-146; Jackson ImmunoResearch Laboratories). Blots were developed with Amersham ECL (RPN2232, Cytiva) chemiluminescent western blot detection reagent and exposed using Amersham Hyperfilm ECL (GE Healthcare).

Cell cultures were grown in triplicate overnight to mid-log phase in YEPD. 10⁷ cells were collected by centrifugation, and protein was extracted by TCA precipitation as described [75 (link)]. Electrophoresis and western blotting were performed as described [76 (link)]. Polyclonal anti-Cdc11 antibodies (sc-7170; Santa Cruz Biotechnology, Dallas, TX, USA) were used at 1:5,000 dilution and monoclonal mouse anti-GFP antibodies (11814460001; Roche, Basel, Switzerland) were used at 1:2,000 dilution. Fluorophore-conjugated secondary antibodies against mouse (IRDye 800CW goat anti-mouse IgG, 926–32210; LI-COR, Lincoln, NE, USA) and rabbit (Alexa Fluor 680 goat anti-rabbit IgG, A21076; Invitrogen, Carlsbad, CA, USA) antibodies were used at 1:10,000 dilution. Blots were visualized and quantified with the ODYSSEY imaging system (LI-COR). All values were normalized to a Cdc11 loading control.