A coimmunoprecipitation assay was performed as described previously (Yoo et al., 2007 (link)). Positive transformants of AOC1‐HA, AOC2‐HA, AOC3‐HA, AOC4‐HA, and GFP‐AN or GFP were lysed in 1 ml of lysis buffer (50 mM Tris‐HCl, pH 7.4, 75 mM NaCl, 5 mM EDTA, 10% glycerol, 0.5% Triton X‐100, 1× protease inhibitor cocktail, 1× phosphatase inhibitor cocktail) and then centrifuged at 2,400 × g for 10 min. For immunoprecipitation, the protein extracts were incubated with GFP agarose beads at 4℃ for 6 h. The immunoprecipitated proteins were washed four times with washing buffer (50 mM Tris‐HCl, pH 7.4, 75 mM NaCl, 5 mM EDTA, 10% glycerol, 0.17% Triton X‐100) before separation by SDS‐PAGE and detection with the respective antibodies (anti‐GFP, Abmart; anti‐HA, Abmart).
Anti gfp
Manufactured by Abmart
Sourced in China, United States
Anti-GFP is a laboratory reagent used to detect and quantify the presence of GFP (Green Fluorescent Protein) in biological samples. It functions as a specific antibody that binds to and identifies GFP, allowing for its visualization and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Anti myc, by Abmart (11 mentions)
Anti flag, by Merck Group (11 mentions)
Anti ha, by Abmart (8 mentions)
Anti flag, by Abmart (5 mentions)
Anti myc, by Merck Group (5 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Anti gst, by Abmart (4 mentions)
Anti actin, by Abmart (4 mentions)
Anti actin, by Merck Group (4 mentions)
Anti myc antibody, by Merck Group (4 mentions)
Anti rfp, by Proteintech (3 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Anti ha, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (2 mentions)
Gfp trap beads, by Proteintech (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Eecl western blot kit, by CWBIO (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
54 protocols using anti gfp
1
Coimmunoprecipitation Assay for Protein Interactions
2
Protein Expression and Western Blot Analysis
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HEK293F cells were transfected with same amounts of plasmids of C-terminal GFP tagged NALCN (WT/mutant)–FAM155A–UNC79–UNC80 in the same ratio for protein expression. Cells were lysed 48 h post transfection in RIPA buffer (Solarbio) with 1.3 μg/mL aprotinin, 0.7 μg/mL pepstatin, 5 μg/mL leupeptin, and 2 mM phenylmethylsulfonyl fluoride (PMSF), followed by sonication. Lysates normalized by the expression level of GAPDH were loaded onto 14% sodium dodecyl sulfate–polyacrylamide gels for electrophoresis (SDS–PAGE). The separated proteins were then transferred to poly-vinylidene fluoride (PVDF) membranes (Merck Millipore). After blocking with 3% BSA for 1 h at 37 °C in Tris-buffered saline with Tween-20 (TBS-T), the membranes were incubated with the primary antibodies for 1 h at 37 °C and then with the horseradish peroxidase (HRP) labeled secondary antibody (1:5000 dilution, CWBIO) for 1 h. WB bands were visualized with the eECL western blot kit (CWBIO). The primary antibodies used were anti-GFP (1:3000 dilution, Abmart) and anti-GAPDH (1:5000 dilution, Proteintech).
3
Antibody Reagents for Protein Analysis
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Anti-Flag (F3165), anti-PARP12 (HPA003584), and anti-FHL2 (HPA006028) antibodies were purchased from Sigma. Anti-HA (ab18181) and anti-GAPDH (ab8245) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-snail (3879S), anti-N-cadhenrin (14215S), and anti-vimentin (5741S) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-GST, anti-His, and anti-GFP antibodies were obtained from Abmart. Anti-(ADP-ribose) antibody (A01316) was purchased from GenScript
Glutathione Sepharose and Ni Sepharose were purchased from GE Heathcare (Little Chalfont, UK, EU). High-capacity Streptavidin Agarose (20359) and S-protein Agarose (69704) were bought from Thermo Scientific and Novagen, respectively.
Glutathione Sepharose and Ni Sepharose were purchased from GE Heathcare (Little Chalfont, UK, EU). High-capacity Streptavidin Agarose (20359) and S-protein Agarose (69704) were bought from Thermo Scientific and Novagen, respectively.
4
Immunoblotting of Redox-Sensitive Proteins
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Cell lysates were prepared using high KCl lysis buffer containing 10 mMTris-HCl, pH 8.0, 140 mMNaCl, 300 mMKCl, 1 mM EDTA, 0.5% TritonX-100 and 0.5% sodium deoxycholate with complete protease inhibitor cocktail (Roche) and 20 mM N-ethylmaleimide (NEMI) to block free thiol groups. Equal amounts of proteins (30 μg) were subjected to SDS-PAGE with loading buffer with or without β-mercaptoethanol and transferred to polyvinylidene fluoride (PVDF) membranes (Roche). The membranes were treated with 1% blocking solution in TBS for 1 h, immunoblots were probed with the indicated antibodies: anti-GFP (1:5,000; AbMart, Shanghai, China), anti-Flag (1:5,000; AbMart, Shanghai, China), anti-HA (1:5,000; AbMart, Shanghai, China), anti-Prdx-SO3 (1:2,000; Abcam) at 4 °C overnight. Then the membranes were washed and incubated with POD-labeled secondary antibodies (1:125,000; Roche). The immunolabeled proteins were detected by BM Chemiluminescence Western Blotting kit (Roche).
5
Immunoblotting of Cell Signaling Proteins
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Human embryonic kidney 293T (HEK293T) cells, HeLa cells, COS-7 cells, and Vero cells were grown in Dulbecco's modified Eagle medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/mL of penicillin and streptomycin at 37°C in a 5% CO2 incubator. Mouse anti-Flag, anti-Myc, anti-GFP, and anti-hemagglutinin (HA) monoclonal antibodies (MAbs) were acquired from Abmart (Berkeley Heights, NJ, USA). IgG negative-control antibody was purchased from Proteintech (Wuhan, China). Rabbit anti-β-actin MAb was bought from ABclonal (Wuhan, China). Rabbit anti-IKKi, anti-TBK1, anti-IRF3, anti-phosphorylated (phospho)-IRF3 (Ser396), and anti-phospho-TBK1 (Ser172) MAbs, alkaline phosphatase (AP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG, and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were provided by Cell Signaling Technology (Boston, MA, USA). Cyanine 5 (Cy5)-conjugated goat anti-rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG were supplied by Bioss (Beijing China) and BBI Life Sciences, respectively.
6
Affinity Purification of MoSWA2-GFP Protein
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The MoARK1‐3xFLAG plasmid containing the hygromycin‐resistance gene was constructed in our previous work (L. Li et al., 2017 ). The MoSWA2 DNA fragment fused with GFP fluorescent protein (MoSWA2‐GFP) was inserted into the pYF11 construct containing the bleomycin resistance gene. The constructs were co‐transformed into wild‐type strain Guy11 and transformants resistant to hygromycin and bleomycin were isolated. Total proteins were extracted from the transformants using protein lysis buffer (50 mM Tris‐HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA and 1% Triton X‐100) and incubated with anti‐Flag® M2 Affinity Gel (Sigma) for 4 h, followed by washing the Affinity Gel with Tris‐buffered saline (TBS) (50 mM Tris‐HCl, 150 mM NaCl, pH 7.4) four times. The proteins that bound to the Affinity Gel were eluted by 0.1 M glycine HCl (pH 3.5) and were detected by anti‐Flag (Sigma‐Aldrich) and anti‐GFP (Abmart) antibodies.
7
Western Blot Protein Detection Protocol
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The separated proteins were transferred from the gel to PVDF membrane and then blocked using PBST (PBS with 0.1% Tween 20) containing 5% non-fat milk for 30 min at room temperature with 60 r.p.m. shaking, anti-GST (1:5000; #M20007; Abmart), anti-his (1:5000; #M30111; Abmart), Anti-Flag (1:5000; #F3165; Sigma-Aldrich), anti-GFP (1:5000; #M20004; Abmart), anti-RFP (1:5000; #5f8; Chromotek), antibodies were added to PBSTM (PBS with 0.1% Tween 20 and 5% non-fat dry milk) and incubated at room temperature for 3–4 h, followed by three times washes with PBST. The membrane was then incubated with a goat anti-mouse IRDye 800CW antibody (Odyssey, no. 926-32210; Li-Cor) at a ratio of 1:10,000 in PBSTM at room temperature for 30 min with 60 r.p.m. shaking. The membrane was washed three times with PBST, and then visualized by excitation at 800 nm. Full-size images are presented in Supplementary Fig. 21 .
8
Poplar Leaf Protein Extraction and Detection
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The crude protein from poplar leaf was extracted using a Plant Protein Extraction Kit (Solarbio, Beijing, China). Total protein concentration was quantified with micro-spectrophotometer ND2000C (Thermo Fisher, Waltham, United States). The tag antibodies, anti-actin, and anti-GFP were purchased from Abmart (Abmart, Shanghai, China) and used according to the manufacturer’s protocol. Immunoblot analysis was carried out by following a previously described protocol (Wang X. et al., 2019 (link)).
9
Co-Immunoprecipitation Assay of VvMYB15 and VvWRKY40
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The Co-IP assay was conducted according to previously described (Lian et al., 2018 (link); Xu et al., 2019 (link)), the cDNA full length of VvMYB15 was cloned and inserted into PHB:Flag to generate PHB:VvMYB15-Flag, the cDNA full length of VvWRKY40 was amplified and inserted into PHB:YFP to generate PHB:VvWRKY40-YFP. A. tumefaciens GV3101 containing the PHB:VvMYB15-Flag was infiltrated into the tobacco leaves alone or together with pHB:VvWRKY40-YFP. The samples were homogenized in lysis buffer [50 mM Tris–HCl, (pH 7.5), 150 mM NaCl, 0.2% Trition-X-100] containing 1 mM Pefabloc and cocktail, and 50 μM MG132. After centrifugation, the supernatant was incubated for 4 h at 4°C with 20 μl of protein G magnetic beads (bed volume, GE Healthcare), which had previously been incubated at 4°C for overnight with 10 μl of anti-GFP antibody (GeneScript). The immunoprecipitates were washed two times with lysis buffer and eluted by boiling with 2 × SDS loading buffer for 5 min. The eluates were subjected to western blot analysis with anti-Flag (Sigma) and anti-GFP (Abmart) antibodies.
10
Protein Interaction Profiling by Co-IP
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Coimmunoprecipitation (Co-IP) assays were performed as described previously, with minor modifications (Shi et al., 2013 (link)). Two days after infiltration, leaves were syringe infiltrated with or without 50 μM of MG132 for 3 h before freezing in liquid nitrogen. For anti-Myc immunoprecipitation, protein extracts were incubated with agarose-conjugated anti-Myc antibody (MBL) for 4 h at 4°C with gentle rotation. The agarose beads were washed and resuspended in 80 μl of PBS and 20 μl of 5 × SDS sample buffer and boiled for 10 min. Protein samples were detected with anti-GFP (Abmart) and anti-Myc (Abmart) immunoblot.
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