Aspergillus nidulans protoplasts were applied to the “Yeast Mitochondria Isolation Kit” (SigmA) and mitochondria isolated as described in the manufacturer’s protocol using detergent lysis (1:200 dilution). In the first centrifugational step, at 600× g mitochondria were obtained in the supernatant (S1). In the second centrifugational step, at 6,500× g mitochondria were sedimented in the pellet (P2). Proteins were analyzed in a Western blot using anti‐GFP antibodies (Roche). For precipitation of the GFP fusion protein (GFP trap), the protein extracts were incubated with the anti‐GFP antibody and protein G agarose (Roche).
Anti gfp antibody
Manufactured by Roche
Sourced in Germany, United States, Switzerland, United Kingdom, Japan
The Anti-GFP antibody is a laboratory reagent designed to detect the presence of the Green Fluorescent Protein (GFP) in biological samples. It is a highly specific and sensitive tool used for the visualization and quantification of GFP-labeled proteins in various experimental settings.
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Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (9 mentions)
Anti flag antibody, by Merck Group (8 mentions)
Protease inhibitor, by Roche (4 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (4 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Protein a g bead, by Santa Cruz Biotechnology (3 mentions)
Fastprep 24, by MP Biomedicals (3 mentions)
Anti myc antibody, by Santa Cruz Biotechnology (3 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Anti actin antibody, by Merck Group (3 mentions)
Anti tubulin antibody, by Merck Group (2 mentions)
Iblot, by Thermo Fisher Scientific (2 mentions)
Anti flag, by Merck Group (2 mentions)
Yeast mitochondria isolation kit, by Merck Group (2 mentions)
Protein g agarose, by Roche (2 mentions)
Complete mini protease inhibitor, by Roche (2 mentions)
Protein g sepharose 4 fast flow, by GE Healthcare (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
148 protocols using anti gfp antibody
1
Mitochondria Isolation from Aspergillus
2
Purification and Detection of Cell Surface Proteins
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Cell surface proteins were purified from transiently transfected HEK293T cells by use of the Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. In brief, cells at 90-95% confluence in a T75 flasks were incubated with 10 ml of sulfo-NHS-biotin solutions (0.25 mg/ml in PBS) for 30 min at 4°C. After the reaction was quenched by 500 µl of quenching solution, cells were scraped, washed with TBS buffer and sonicated. The protein concentration was determined by Bradford Assay. Biotinylated proteins were incubated with Immobilized NeutrAvidin Gel (Pierce), eluted according to the manufacturer's directions and loaded onto SDS-PAGE, and immunoblots were probed with anti-GFP antibodies (1:1000, Roche, 11814460001) to detect GFP-VANGL2. Detection of tubulin with anti-tubulin antibody (1:2500, Sigma-Aldrich, T9026) was used as a threshold above which samples were considered to be contaminated with intracellular proteins. All experiments were done in biological triplicate.
3
Antibodies for Protein Detection
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Anti-GFP antibodies (a mix of clones 7.1 and 13.1 was used) were
purchased from Roche Applied Science. Anti-Renillaluciferase antibodies (mAb4400 and mAb4410) were purchased from
Chemicon. Secondary antibodies coupled to horseradish peroxidase were
purchased from Dako. Alexa-conjugated antibodies for immunostaining are
from Jackson ImmunoResearch laboratories.
purchased from Roche Applied Science. Anti-Renillaluciferase antibodies (mAb4400 and mAb4410) were purchased from
Chemicon. Secondary antibodies coupled to horseradish peroxidase were
purchased from Dako. Alexa-conjugated antibodies for immunostaining are
from Jackson ImmunoResearch laboratories.
4
Quantifying SUMO Modifications in C. elegans
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Worms were collected at different time points in M9 buffer, spun down and equal volume 2x SDS lysis buffer (50 mM NaPO4 pH 7.5, 600 mM NaCl, 2%SDS) was added. Worms were frozen in liquid nitrogen, thawed and sonicated at 10% for 2 min with Branson sonifier (Branson). The lysates were spun down. The protein content was measured with BCA kit (Thermo Scientific). 20 μg of protein lysate was loaded on each lane and separated by SDS-PAGE followed by electroblotting on PVDF membrane. The membrane was blocked with 5% non-fat milk and incubated with anti-GFP antibodies (Roche) o/n followed by horse radish conjugated secondary antibody. Rabbit antibody against C. elegans SUMO was generated against the N-terminal peptide MADDAAQAGDNAEYIKIK (Eurogentec). Antibodies used to confirm the SUMO modification included: anti actin (MAB1501 from Chemicon), anti alpha tubulin clone DM1A (Sigma T9026), anti mannosidase II (AbDSerotec AHP674), anti cytochrome C1 (custom made), anti catalase (SantaCruz sc-365738) anti GRP94 (SantaCruz sc-11402). The secondary antibody was detected with ECL system.
5
Quantitative Analysis of GFP Proteins
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Total proteins of homozygous GFP1 and GFP2 lines, which displayed the phenotypes similar to the OE5 line, were extracted in 20mM Tris-HCl, pH 8.0, 5mM EDTA, 1mM PMSF, 0.05% SDS, 5mM EGTA, and 10mM DTT. After sedimentation at 4 °C for 1h, the solution was centrifuged at 12 000×g for 30min. The supernatants were then used for protein quantification in a Microplate system as described previously (Dreher et al., 2005 (link)). Total proteins were electrophoresed in 10% SDS-PAGE and the gels were transferred onto polyvinylidene fluoride microporous membranes. The membranes were treated with anti-GFP antibodies (Roche, Germany) diluted 1000 fold for 1h at 4 °C, followed by treatment with secondary antibodies (Goat anti-Mouse IgG-HRP, Abmart, China) for 1h.
6
GFP-Cdt 2-345 Affinity Purification
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GFP-Cdt 2–345 (Fig. 1 A ) was immunoprecipitated from lysates of transiently transfected U2OS cells by anti-GFP antibodies (Roche) bound to G/A beads (Santa Cruz Biotechnology, Inc.). After three washes and elution from the beads, GFP-Cdt 2–345 and coprecipitated proteins were subsequently digested with trypsin and Lys-C. Samples were desalted on C-18 stage tips (Nest Group) and analyzed by nanoflow HPLC tandem mass spectrometry (2D-NanoLC; Eksigent; Orbitrap Velos mass spectrometry; Thermo Fisher Scientific; Vasilj et al., 2012 (link)). Identification of proteins was performed with Mascot (V2.2; Matrix Science), and isoform specificity of peptides was obtained from the Proteomicsdb database (Wilhelm et al., 2014 (link)).
7
Measuring Ribophagy Using GFP Fusion Proteins
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Cells grown in YPD or synthetic medium were collected during the exponential growth phase (OD600 of 1.5 or 0.8, respectively). Total protein extracts were prepared by the NaOH-TCA lysis method and analyzed by Western blotting using anti-GFP antibodies (Roche). Signals corresponding to GFP fusion proteins and GFP were quantified using the ImageJ software (National Institutes of Health), and the ratio of cleaved to noncleaved GFP protein was used to measure the ribophagy process (Kraft et al., 2008 (link); Ossareh-Nazari et al., 2010 (link)).
8
Protein Extraction and Analysis from Yeast
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Cells were grown to mid-logarithmic phase in SC at 30°C, washed three times with SD-N medium, and incubated for 3 h in SD-N at 30°C. At the indicated times, 3 OD600 units of cells were withdrawn. The cells were pelleted, resuspended in 50 µl of 1.85 M NaOH, incubated on ice for 10 min, mixed with 125 µl of 20% trichloroacetic acid, and incubated on ice for 30 min. The precipitated proteins were separated by SDS-PAGE and immunoblotted with anti-GFP antibodies (Roche).
9
Western Blot Protein Analysis
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Protein samples were prepared by boiling the pelleted cells in radioimmunoprecipitation assay buffer, as described (41 (link)). The membranes were probed with 1:5000 mouse monoclonal anti-HA (16B12; Covance), 1:5000 rabbit polyclonal anti-FRB (from David Shore, University of Geneva), 1:5000 rabbit polyclonal anti-RAP1 (from David Shore, University of Geneva), 1:10,000 rabbit polyclonal anti–glyceraldehyde-3-phosphate dehydrogenase (A9521; Sigma-Aldrich), and 1:5000 mouse monoclonal anti-GFP antibodies (11814460001, Roche). Horseradish peroxidase–conjugated goat anti-mouse (Bio-Rad) or anti-rabbit (A8275, Sigma-Aldrich) immunoglobulin G were used at 1:10,000 as secondary antibodies.
10
Analyzing GOT-1::GFP in C. elegans
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Mixed stage C. elegans were grown on 100 mm NGM plates seeded with OP50 and collected using M9 buffer. Lysates were prepared as described previously [78 (link)]. For western blot analyses, total protein concentration was determined using a Bradford Protein Assay (Bio-Rad). Anti-GFP antibodies (Roche) were used to probe for GOT-1::GFP and tubulin was used for the loading control.
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