BZR1 fused to GST was purified from bacteria using glutathione beads (GE Healthcare, London, UK). EBF1 fused to MBP were purified using amylose resin (New England Biolabs, Ipswich, MA, USA). glutathione beads containing 1 µg of GST-BZR1 were incubated with 1 µg MBP or MBP-EBF1 as indicated by the pull-down buffer (20 mM Tris-HCl pH7.5, 100 mM NaCl, 1 mM EDTA), and the beads were washed 10 times with the wash buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.5% TritonX-100, 1 mM EDTA). The eluted proteins were analyzed by immunoblot analysis with an anti-MBP antibody (New England Biolabs, Beijing, China, Cat: E8038L, 1:5000 dilution).
Anti mbp antibody
Manufactured by New England Biolabs
Sourced in United States, China
The Anti-MBP antibody is a highly specific and sensitive reagent designed for the detection and analysis of maltose binding protein (MBP) fusion proteins. It is a polyclonal antibody produced in rabbits and purified by affinity chromatography. The antibody recognizes the MBP portion of fusion proteins and can be used in various immunological applications, such as Western blotting, ELISA, and immunoprecipitation.
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Lab products found in correlation
Amylose resin, by New England Biolabs (6 mentions)
Anti ubiquitin antibody, by Santa Cruz Biotechnology (3 mentions)
Ni nta agarose, by Qiagen (3 mentions)
Glutathione beads, by GE Healthcare (2 mentions)
Recombinant human h5b, by Enzo Life Sciences (2 mentions)
Creatine kinase, by Merck Group (2 mentions)
Bovine ubiquitin, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti caga, by Santa Cruz Biotechnology (1 mentions)
Clarity ecl, by Bio-Rad (1 mentions)
Quick start bradford assay, by Bio-Rad (1 mentions)
Amersham hyperfilm ecl, by GE Healthcare (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Hrp conjugated rabbit anti chicken igy, by Merck Group (1 mentions)
Anti gst, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (1 mentions)
Hispur ni nta resin, by Thermo Fisher Scientific (1 mentions)
29 protocols using anti mbp antibody
1
GST-BZR1 and MBP-EBF1 Binding Assay
2
Lipid-Protein Binding Assay for Kv7 Channels
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Lipid–protein overlay assay was performed according to the protocol (39 (link)). Briefly, the plasmids inserted with the C-terminus of the WT or SUMO2-fusion Kv7.2 and Kv7.3 were constructed, and E. coli BL21 competent cells containing plasmids were grown in growth media with IPTG. The proteins containing MBP tag were purified with the chromatography column packed with amylose resin and concentrated. PIP strips were 2 × 6 cm hydrophobic membranes that have been spotted with 100 pM of all eight phosphoinositides and seven other biological important lipids (Echelon biosciences). PIP strips were blocked with 3% bovine serum albumin (Sigma) and gently agitated for 1 h at RT. The blocking buffer was discarded, and the strips were incubated with equal purified proteins overnight at 4 °C with gentle agitation. The strips were washed and incubated with anti-MBP antibody (NEB), followed by a secondary antibody coupled to peroxidase, and the interacted protein levels were detected with a chemo-luminescence system (Tanon) after additional washing steps.
3
Polyclonal Antibodies Against Cag Proteins
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Polyclonal antibodies against Cag proteins such as Cagδ, CagH, CagL, CagE, CagY, CagZ, CagX, CagT, CagM, CagI, and CagF were raised by immunization of rabbit and mice in the laboratory as described previously 14 (link), 29 (link). Briefly, to generate polyclonal antibody against CagV, a full‐length cagV was cloned in pET‐28a vector and overexpressed in E. coli strain BL‐21 (DE3). CagV protein band was excised from SDS/PAGE, used for immunization, specificity and titer of rabbit anti‐CagV (α‐CagVr) and mice anti‐CagV antibody (α‐CagVm) were tested by western blotting. Anti‐CagA, anti‐OMP (Outer Membrane Protein), and anti‐HSP (Heat Shock Protein) antibodies were from Santa Cruz, CA, USA (Cat. No. Sc‐25766, Sc‐69935 and Sc‐57779, respectively). Anti‐MBP antibody was from NEB, USA (Cat. No. E8032L). SDS/PAGE and western blotting were performed as described previously 14 (link), 29 (link).
4
Protein Redox State Profiling
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BIAM labeling and biotin-switch assays were performed as described previously (Tian et al., 2018 (link)). For BIAM labeling, purified MBP-OsRPL38 proteins were treated with different concentrations of H2O2 at room temperature for 20 min in the dark. Proteins labeled with BIAM were separated on a 12% SDS–PAGE gel and detected with anti-biotin and horseradish peroxidase-linked antibodies (1:5000 dilution, Cell Signaling Technology, USA). Input MBP or MBP-OsRPL38 proteins were detected using an anti-MBP antibody (1:5000 dilution, New England Biolabs, USA).
5
Recombinant Protein Characterization Protocol
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Protein concentration was determined using the QuickStart Bradford Assay (BioRad, UK), following the manufacturer's protocol. Recombinant proteins were transferred to polyvinylidene difluoride (PVDF) membrane using the TransBlot Turbo system (Biorad, UK) and analysed by Western blot with anti-GST (Santa Cruz Biotech, USA) or anti-MBP antibody (New England Biolabs, UK) at 1:10,000 dilutions. Bound antibodies were detected using appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies at 1:10,000 dilutions. To assess if the cloned antigens were immunogenic following natural Campylobacter infection, pooled serum from Campylobacter-infected non-vaccinated White Leghorn birds collected three weeks post-infection was used at a 1:100 dilution. Bound serum IgY was detected with an HRP-conjugated rabbit-anti chicken IgY (Sigma-Aldrich, UK) at a 1:3000 dilution. In order to analyse the subcellular localisation of SodB a Western blot of subcellular fractions (Section 2.9 ) was probed with sera from GST–SodB vaccinated birds as above. Blots were developed using Clarity ECL (BioRad, UK) and autoradiography (Amersham Hyperfilm ECL, GE Lifesciences, UK).
6
Protein-Protein Interaction Assay
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Recombinant MBP-NbWRKY40, MBP-NbWRKY8, or MBP alone from E. coli lysates were immobilized on amylose resins (NEB), incubated for 1 h at 4°C with purified GST-XopS or GST alone, eluted, and analyzed by immunoblotting using either anti-GST antibody (1:1,000, horseradish peroxidase-conjugated; Santa Cruz Biotechnology Inc.; cat. no. sc-138 HRP) or anti-MBP antibody (1:10,000; NEB, cat. no. E8032S; used with 1:10,000 secondary horseradish peroxidase-conjugated anti-mouse antibody; ThermoFisher; cat. no. 31430).
7
MBP-CaDTR1 Ubiquitination Assay Protocol
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The procedure for the expression and purification of the maltose-binding protein (MBP)–CaDTR1 recombinant protein is described in Park et al.24 (link). For the in vitro ubiquitination assay, the purified MBP–CaDTR1 (500 ng) was mixed with ubiquitination reaction buffer [50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.05 mM ZnCl2, 1 mM Mg-ATP, 0.2 mM DTT, 10 mM phosphocreatine, and 0.1 unit of creatine kinase (Sigma-Aldrich)] containing 250 ng of recombinant human UBE1 (Boston Biochemicals, Cambridge, MA, USA), 250 ng of recombinant human H5b (Enzo Life Sciences, Farmingdale, NY), and 10 μg of bovine ubiquitin (Sigma-Aldrich). After incubation at 30 °C for 3 h, the reacted proteins were separated using SDS-PAGE and analysed using immunoblotting with anti-ubiquitin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-MBP antibody (New England Biolabs, Ipswich, MA).
8
In vitro Degradation of AtACS7
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Ten-day-old light-grown seedings were harvested and ground in liquid nitrogen to a fine powder. Three volumes of in vitro degradation buffer (25 mM Tris–HCl pH 7.5, 10 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 5 mM dithiothreitol, and 10 mM ATP) were added and lysed on ice for 30 min. After centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatants were incubated with 1.5 μg MBP-AtACS7-His or MBP-AtACS7K285RK366R-His at 28 °C for the indicated times. Reactions were blocked by adding a sample buffer (250 mM Tris-HCl pH 6.8, 50% glycerol, 10% SDS, 0.5% bromophenol blue, 5% β-mercaptoethanol). Then, the samples were separated by 12% SDS-PAGE and transferred to a PVDF membrane. The AtACS7 protein was detected using an anti-MBP antibody (NEB, Beijing, China) and PAG1 was detected using an anti-PAG1 antibody (Abcam, Cambridge, UK). Image J (http://rsb.info.nih.gov/ij/ , last accessed 19 February 2024) software was used for quantification of the protein band intensities from immunoblots.
9
MBP-CaREL1 Ubiquitination Assay
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The procedure for the expression and purification of the maltose-binding protein (MBP)–CaREL1 recombinant protein is described in Park et al.42 (link). For the in vitro ubiquitination assay, the purified MBP–CaREL1 (500 ng) was mixed with ubiquitination reaction buffer [50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.05 mM ZnCl2, 1 mM Mg-ATP, 0.2 mM DTT, 10 mM phosphocreatine, and 0.1 unit of creatine kinase (Sigma-Aldrich)] containing 250 ng of recombinant human UBE1 (Boston Biochemicals, Cambridge, MA, USA), 250 ng of recombinant human H5b (Enzo Life Sciences, Farmingdale, NY), and 10 μg of bovine ubiquitin (Sigma-Aldrich). After incubation at 30 °C for 3 h, the reacted proteins were separated using SDS-PAGE and analysed using immunoblotting with anti-ubiquitin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-MBP antibody (New England Biolabs, Ipswich, MA).
10
FGFR2 Domain Combinations via PIPE Cloning
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Polymerase incomplete primer extension (PIPE) cloning was used to obtain specific domain combinations of FGFR2, and the cloning vector pSpeedET with an N-terminal E. coli maltose binding protein (MBP) fusion tag of 42.5 kDa (Klock & Lesley, 2009 (link)). The domain combinations created are shown in Table 1 . The FGFR inserts were amplified by PCR using Phusion Hi Fidelity DNA Polymerase, 200 mM dNTP, 0.5 µM forward and reverse primers, and 6% DMSO. PCR products were extracted from agarose gel and purified using Thermo Scientific GeneJet Gel Extraction Kits. The MBP fusion tag was added to the construct to improve construct solubility and expression (Kapust & Waugh, 1999 (link)), allow purification by amylose affinity chromatography, and identification by Western blot with an anti-MBP antibody (New England Biolabs (E-8038)). Cloning results were confirmed by DNA sequencing.
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