The total protein concentration of cell lysates was determined spectrophotometrically via Bradford assay. The total protein concentration of the lysates used in this work is controlled to be within the range of 5 to 10 mg mL−1. To qualify our specific proteins of interest tagged with GFP, the cell lysates were analyzed by western blotting. The samples were stained with rabbit anti-GFP (Millipore Sigma) and mouse anti-GAPDH anti-bodies (Invitrogen) followed by secondary staining with goat anti-rabbit IgG Alexa Fluor 488 and goat anti-mouse Alexa Fluor 647 (Invitrogen).
Rabbit anti gfp
Manufactured by Merck Group
Sourced in United States, Germany
Rabbit anti-GFP is a laboratory reagent used to detect and quantify green fluorescent protein (GFP) in various biological samples. It is a polyclonal antibody produced by immunizing rabbits with GFP. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and flow cytometry to identify and measure GFP-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti flag, by Merck Group (4 mentions)
Rabbit anti flag, by Merck Group (3 mentions)
Mouse anti myc, by Merck Group (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Mouse anti α tubulin, by Merck Group (2 mentions)
Irdye 800cw secondary antibody, by LI COR (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Bioruptor, by Diagenode (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Rabbit anti myc, by Merck Group (2 mentions)
Rabbit anti protein a, by Merck Group (2 mentions)
Mouse anti gfp, by Merck Group (2 mentions)
Mouse anti flag m2 antibody, by Merck Group (2 mentions)
Mouse anti ha, by Merck Group (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Mouse anti ha, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Mouse anti gapdh antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
35 protocols using rabbit anti gfp
1
Protein Quantification and Verification
2
Immunofluorescence Analysis of Humanized Mouse Thymus
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Half of the thymic graft from each humanized mouse was fixed in 4% paraformaldehyde for 30 min at room temperature, then transferred to 30% glucose PBS at 4°C overnight. Tissues were then embedded in OCT compound and frozen. Cryosections (5 μm) were blocked with goat serum (Vector Laboratories) for 15 min, and stained with following primary antibodies: rabbit anti-GFP (Millipore/Sigma) and mouse anti-Pan-cytokeratin (Abcam) in room temperature for 3 h or 4°C overnight. Sections were subsequently stained with AF488 rabbit anti-GFP IgG (H+L) antibody (Jackson ImmunoResearch) and AF647 goat anti-mouse IgG (H+L) antibody (Jackson ImmunoResearch) at room temperature for 1 h. All sections were mounted in mounting medium with DAPI (Vector Laboratories). Images were obtained using Leica DMI 6000B wide field microscope.
3
Immunoprecipitation and Western Blot Analysis
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Cell lysates were incubated with protein A/G-agarose beads (Millipore) for 1 h at 4 °C to minimize nonspecific binding. Lysates were transferred to clean tubes and incubated further with the appropriate antibody, including rabbit anti-GFP or rabbit anti-Flag (Sigma). After incubation at 4 °C overnight, protein A/G-agarose beads were added and rotated for 2 h. The complexes were washed three times with 1× immunoprecipitation buffer. Proteins were eluted by boiling in 2× SDS loading buffer and subjected to western blot using GFP, Flag, p-Paxillin, and Paxillin antibodies.
4
Quantitative Western Blot Analysis
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Cells were harvested 48 hr after transfection and lysed in STEN lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% SDS, 1% NP-40, and 2 mM EDTA + protease inhibitor cocktail (Roche)) by sonicating for 5 min at medium power using a Bioruptor (Diagenode), followed by 15-min incubation on ice. Protein concentration was measured using the Bradford assay (Bio-Rad) with BSA as a standard. Rabbit anti-GFP (Sigma G1544) and mouse anti-α-tubulin (Sigma T9026) antibodies were used with IRDye 800CW secondary antibodies (LI-COR) for Western blotting, and the blots were imaged and quantified using the Odyssey Infrared Imaging System (LI-COR).
5
Antibody Characterization for Cell Biology
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The following primary antibodies were used at a dilution of 1:1000: rabbit anti-APC2 (Cell Signaling Technology, CST12301), rabbit anti-CUL4A (Bethyl, A300-739A), rabbit anti-APC4 (Bethyl, A301-176A), rabbit anti-V5 (Abcam, ab9116), mouse anti-SUMO2/3 (Abcam, ab81371), mouse anti-SUMO2/3 (MBL-Sanbio, M114-3), rabbit anti-GFP (Sigma, 1814460), mouse anti-His (Sigma, H1029), rabbit anti-SART1 (custom-made by Eurogentec)67 (link), rabbit anti-CDC27 (Santa Cruz, sc9972), rabbit anti-CDH1, rabbit anti-Hsl1 and rabbit anti-APC11 (kind gifts from Dr. J.M. Peters, Vienna)66 (link), rabbit anti-KIF18B (Bethyl, A303-982A), and rabbit anti-Securin (Cell Signaling Technology, CST13445).
6
Immunostaining of Cryosectioned Samples
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Cryosections were blocked for 1 hour in 2% BSA in TBST and incubated in primary antibody at room temperature overnight. The sections were washed three times in 4% milk in TBST for 5 min each, followed by 1 hour incubation with rabbit anti-GFP (1/500) (Sigma-Aldrich Chemical Co., St. Louis, MO), FITC-labeled goat anti-rabbit secondary (1/1000) (Sigma-Aldrich Chemical Co., St. Louis, MO), TRITC-conjugated phalloidin (1/40) (Sigma-Aldrich Chemical Co., St. Louis, MO), and 264 μM Hoechst 33258 with appropriate washes in between. Slides were visualized with a Zeiss Axioobserver Z1 equipped with an AxioCam MRm controlled by AxioVision4.6 software.
7
Antibody Validation for Molecular Signaling
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All antibodies were from commercial sources: HA—horseradish peroxidase (anti-HA-HRP), 3F10 (Roche, Basel, Switzerland), rabbit monoclonal anti-MST2 (Abcam), goat polyclonal anti-MST2 (C-19; Santa Cruz, CA, USA), rabbit polyclonal p-T180-MST2 (Cell Signaling, Danvers, MA, USA), goat polyclonal anti-LATS1 (n-18 and g-16; Santa Cruz), rabbit monoclonal p-T1079-LATS1, rabbit polyclonal anti-YAP1 (Santa Cruz), mouse monoclonal anti-YAP1 (Sigma, Dallas, TX, USA), rabbit polyclonal p-S127-YAP (Cell Signaling), mouse monoclonal anti-IQGAP1 (MBL), rabbit polyclonal anti-IQGAP1 (Santa Cruz), mouse monoclonal C-Myc tag (Santa Cruz), AKT, p-S308-AKT, p-S473-AKT (Cell Signaling), mouse monoclonal anti-Tubulin and rabbit monoclonal anti-GAPDH (Santa Cruz), mouse monoclonal anti-FLAG M2-Peroxidase (Sigma), rabbit monoclonal anti-GFP, mouse monoclonal anti-TEF-1 (BD Biosciences), mouse monoclonal anti-p-T183/Y185-ERK1/2 and rabbit polyclonal anti-ERK1/2 (Sigma). Mouse monoclonal anti-GFP (Roche), rabbit anti-GFP or goat antiserum (Sigma-Aldrich, St Louis, MO, USA) were used as isotopic IgG immunoprecipitation control.
8
Brains Fixed and Immunostained
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Brains were removed from the head casing and fixed in 4% paraformaldehyde in phosphate buffer solution (PBS; 1.86 mM NaHPO, 8.41 mM NaHPO, and 175 mM NaCl) for 1 h and washed in PBS. Following a 2-h pre-incubation in 3% normal goat serum in PBS-TX (PBS containing 0.3% Triton X-100), brains were washed in PBS-TX. Primary antibodies were Rabbit anti-GFP (1:1000; Sigma) and mouse anti-PDF (1:50; DSHB, University of Iowa), washed in PBS-TX and incubated in the appropriate fluorescent secondary antibodies.
9
Immunostaining of Drosophila CNS
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The central nervous system (CNS) was dissected from flies in PBS, and fixed in 4% paraformaldehyde dissolved in PBS for 60 min. Immunostaining was carried out as described previously [16 (link)], using the following antibodies at the indicated dilutions: rabbit anti-GFP (Sigma, A6455; 1:500), mouse Anti-Brp (nc82) (DSHB, 1:10), guinea pig anti-FruMale [48 (link)] (1:500) and rabbit anti-Camta (this study, 1:200). The secondary antibodies used were as follows: Alexa488-conjugated goat anti-rabbit IgG (Thermo Fisher, A-11034) at 1:500, Alexa546-conjugated goat anti-mouse, rabbit or guinea pig IgG (Thermo Fisher, A-11003, A-11035 or A-11074) at 1:500, and Alexa647-conjugated goat anti-mouse IgG (Thermo Fisher, A32723) at 1:500. Stacks of optical sections were obtained with a Zeiss LSM 510 META confocal microscope and were processed with Image J software.
10
Kinesin Protein Expression and Detection
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HEK 293T cells or DRG sensory neurons were collected and prepared with lysis buffer (1% NP-40; 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 2 mM EDTA; protease inhibitor [Sigma-Aldrich]; and phosphatase inhibitor [Life Technologies]). Cell lysates were placed on ice for 20 min and centrifuged at 13,000 rpm for 20 min to collect the supernatant. Lysates were separated with 4–12% Bis-Tris NuPAGE gel (Thermo Fisher Scientific) and blotted with the following primary antibodies: mouse anti-SFPQ (1:1,000; Sigma-Aldrich), rabbit anti-KIF5A (1:2,000; Abcam), rabbit anti-KIF5B (1:2,000; Abcam), rabbit anti-KIF5C (1:2,000; Abcam), rabbit anti-KIF3C (1:500; Abcam), rabbit anti-KIF3A (1:200; Abcam), rabbit anti-KIF1A (1:5,000; Abcam), mouse anti-HAlo (1:1,000), rabbit anti-KLC1 (1:500; Santa Cruz Biotechnology), rabbit anti-KLC2 (1:1,000; Proteintech), rabbit anti-pan actin (1:1,000; Cell Signaling Technologies), mouse anti-Myc (1:500; Santa Cruz Biotechnology), mouse anti-FLAG (1:1,000; Sigma-Aldrich), mouse anti-HA (1:10,000; Thermo Fisher Scientific), and rabbit anti-GFP (1:2m000; Sigma-Aldrich). HRP-conjugated secondary antibodies (1:10,000; BioRad), ECL detection system (VWR International), and SuperSignal West Dura (Thermo Fisher Scientific) were used for chemiluminescent detection.
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