Livers were lysed with RIPA buffer (9806; Cell Signaling) containing 1 mM phenylmethylsulfonyl fluoride (8553S; Cell Signaling). The protein concentration of each sample was determined using a bicinchoninic acid (BCA) assay reagent (Vigorous Biotechnology) according to the manufacturer’s recommendations. An equal amount of each protein sample was electrophoresed on a 10% acrylamide gel and the bands were then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The membrane was blocked with 5% non-fat dry milk for 3 h and incubated with GFP antibody (#2555; Cell Signaling) and internal control β-actin antibody (ab8227; Abcam) overnight at 4°. The PVDF membrane was then washed three times for 30 min in 0.1% Tween-20 in Tris-buffered saline (TBST) and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zhongshan). After washing for 30 min with three changes of TBST, the membrane was treated with the Pierce ECL 2 western blotting Substrate (Thermo Scientific).
Gfp antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The GFP antibody is a laboratory tool used to detect and visualize the presence of green fluorescent protein (GFP) in biological samples. It functions by specifically binding to GFP, allowing researchers to identify and localize GFP-tagged proteins within cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Internal control β actin antibody, by Abcam (1 mentions)
Pierce ecl 2 western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ecl plus kit, by Merck Group (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Cytochrome c antibody, by BD (1 mentions)
Ab125096, by Abcam (1 mentions)
Mouse monoclonal mcherry antibody, by Abcam (1 mentions)
Alexa fluor 647 goat anti mouse igg h l antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l antibody, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Hrp conjugated anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Las 3000 system, by Fujifilm (1 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
11 protocols using gfp antibody
1
Quantitative Western Blot Analysis of GFP
2
Western Blot Analysis of GFP and His-Tagged Proteins
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Samples from the same number of bacterial cells were loaded onto 10 or 12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel. Then the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane and probed with a GFP antibody or a mouse monoclonal antibody against the 6 × His tag (1:2000; Cell Signaling Technology, United States) at room temperature for 1–2 h or overnight at 4∘C. Then the PVDF membrane was washed with 1 × phosphate-buffered saline (1 × PBS, 5.4 mM KCl, 20 mM Na2HPO4, 274 mM NaCl, 4 mM KH2PO4, pH 7.4) containing 2% 24 times. Next, the PVDF membrane was incubated with an anti-rabbit IgG (1: 2,000; Promega, United States) at room temperature for 1.5 h. Signals were detected by an ECL Plus kit (Millipore). The signals were visualized by a Bio-Rad molecular imager (ChemiDocXRS). The RNA polymerase α subunit RpoA was used as a loading control (with an antibody from Biolegend).
3
Tracking Infused Cells in Mice
4
CRY1 Interaction with Autophagy
5
Immunodetection of cellular proteins
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Primary antibody sources were as follows: mouse monoclonal mCherry antibody (diluted 1:1,000 for western blotting, 1:50 for EM, ab125096), rabbit monoclonal CD9 antibody (diluted 1:50 for EM, ab92726) and NF-κB antibodies (diluted 1:1,000 for immunocytochemistry, ab16502) were obtained from Abcam (Cambridge, MA, USA); CD63 (diluted 1:1,000 for western blotting, sc-15363) and GAPDH (diluted 1:4,000 for western blotting, sc-25778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); cytochrome c antibody (diluted 1:1,000 for immunocytochemistry, 556432) was from BD biosciences (San Jose, CA, USA); and the GFP antibody (diluted 1:1,000 for western blotting, 2555) was from Cell Signaling Technology (Beverly, MA, USA). Secondary antibody sources were as follows: anti-rabbit (sc-2004) and mouse secondary antibody (sc-2005) were obtained from Santa Cruz Biotechnology, and Alexa Fluor 647 goat anti-mouse IgG (H+L) antibody (diluted 1:1,000 for ICC, A-21235) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody (diluted 1:1,000 for ICC, A-11008) were purchased from Thermo Fisher Scientific (Wilmington, DE, USA).
6
Kidney Protein Extraction and Western Blotting
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Kidney tissues were lysed in RIPA buffer with protease inhibitors (0.5 mm PMSF and 1x cOmplete Protease Inhibitor Cocktail, Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitors (50 mM NaF, 1.5 mM Na3VO3), and the lysates were mixed with Laemmli buffer for SDS-PAGE for western blotting analyses. Cultured cells and urine samples were lysed directly in 1x Laemmli buffer containing protease inhibitors and phosphatase inhibitors. Anti-β-actin antibody was from Sigma (A5441, 1:5,000; St. Louis, MO), ClC-5 Rabbit polyclonal antibody from GeneTex (GTX53963, 1:500, Irvine, CA), CC16 rabbit polyclonal antibody from BioVendor (RD181022220-01, 1:500, Asheville, NC), albumin goat polyclonal antibody from Bethyl Laboratories (A80-129A, 1:1,000, Montgomery, TX), DBP rabbit polyclonal antibody from Proteintech (16922-1-AP, 1:1,000, Rosemont, IL), megalin rabbit polyclonal antibody from Abcam (ab76969, Boston, MA), and GFP antibody was from Cell Signaling Technology (Danvers, MA, cat. no. 2555S). HRP-conjugated anti-mouse IgG (H + L) (Thermo Fisher Scientific, cat. no. 31430, 1:5,000) and anti-rabbit IgG (H + L) (cat. no. 31460, 1:5,000) secondary antibodies were used in western blotting. Chemiluminescence reagents (Thermo Fisher Scientific) were used to visualize the protein signals under an LAS-3000 system (Fujifilm, Tokyo, Japan).
7
RNA-Binding Protein Immunoprecipitation
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According to the manufacturer's instructions, RNA immunoprecipitation (RIP) experiments were accomplished with a Magna RIP RNA‐Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA). GFP antibody was used for RIP (Cell Signalling Technology, #55494). Coprecipitated RNA was estimated by qRT‐PCR assay.
8
Antibody Immunoblotting Protocol
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Primary antibody sources: mCherry antibody (#43590), GFP antibody (#2956), FKBP1A/FKBP12 antibody (#55104), CD9 antibody (#13174), TSG101 antibody (#72312), and Alix antibodies (#18269) were diluted 1:1000 for western blotting, which was purchased from Cell Signaling Technology (Beverly, MA, USA); mTOR (human FRB Domain) antibody (diluted 1:1000 for WB, ALX-215-065) was procured from Enzo Life Science (Farmingdale, NY, USA); HSP70 antibody (diluted 1:1000 for WB, #242707) was obtained from R&D systems (Minneapolis, MN, USA); Beta actin antibody (diluted 1:1000 for WB, ab13772) was purchased from Abcam (Cambridge, MA, USA). Secondary antibody sources: anti-rabbit (#32460) and anti-mouse secondary antibody (#32430) were obtained from Thermo Fisher Scientific (Wilmington, DE, USA).
9
Signaling Pathway Activation Assay
10
Preclinical Evaluation of Tumor Metastasis
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The animal study was approved by the Ethical Committee of Xiangya Hospital, Central South University. At least 6 nude mice per group were used to ensure the adequate power, and mice were randomly allocated to the groups. For tail-vein injection, 1−2 × 106 cells in 100 μL PBS were injected into the lateral tail vein of BALB/c-nu mice (4−6 weeks old; 18−20 g). Mice were monitored twice weekly and were sacrificed by cervical dislocation depending on their survival time. Lungs of each group of mice were dissected and fixed with 4% paraformaldehyde and subjected to H&E staining. The tumor cell number was evaluated in 9 random fields (square millimeters) of the H&E tissues under 20× magnification using M8 Digital Microscopy (PreciPoint). To detect CTCs in the animal samples, 100 μL whole-blood samples were collected by tail-vein bleeding into EDTA-coated tubes 14 days after intravenous (i.v.) injection. GFP-positive tumor cells were selected by flow cytometry using GFP antibody (Cell Signaling Technology), and viable cells were counted using trypan blue exclusion assay.
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