To detect the binding of MRP to immobilized hFg, 96-well plates were coated overnight at 4 °C with 100 μl of 2.5 μg/ml Fg/Fg-D/Fg-E (Merck). After blocking with 3% BSA (bovine serum albumin) in PBST (phosphate-buffered saline with 0.05% Tween-20), recombinant MRP proteins were added and incubated for 1 h at room temperature. Bound MRP was detected by incubation with an anti-his monoclonal antibody (1:5000, Sigma) and HRP (horseradish peroxidase)-conjugated anti-mouse antibodies (1:10,000; Santa Cruz). The binding was quantified by measuring the absorbance at 450 nm. Background values obtained for the control (incubated with PBST containing 1% BSA, without truncated MRP) were subtracted. To detect the binding of hFg to immobilized MRP, 96-well plates were coated overnight at 4 °C with 100 μl of 100 nM truncated MRP. After blocking with 3% BSA in PBST, biotinylated hFg was added, and the plates were incubated for 1 h at room temperature. Bound hFg was detected by incubation with HRP-conjugated streptavidin (1:8000; GE Health). Background values obtained for the control (incubated with PBST containing 1% BSA, without biotinylated hFg) were subtracted. The assays were performed in triplicate, and the mean ± SD is shown.
Anti his monoclonal antibody
Manufactured by Merck Group
Sourced in United States, Germany, Macao
The Anti-His monoclonal antibody is a laboratory reagent used for the detection and purification of recombinant proteins containing a histidine (His) tag. It specifically binds to the His-tag sequence, allowing for the identification and isolation of the target protein from complex mixtures.
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Lab products found in correlation
Reduced glutathione, by Merck Group (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Complete edta free protease inhibitor, by Roche (2 mentions)
Hrp conjugated streptavidin, by GE Healthcare (1 mentions)
Dh10bac, by Thermo Fisher Scientific (1 mentions)
Ncoi and hindiii, by New England Biolabs (1 mentions)
Nickel nta beads, by Qiagen (1 mentions)
Pgem 3z, by Thermo Fisher Scientific (1 mentions)
Pfastbachtb, by Thermo Fisher Scientific (1 mentions)
Prsetb, by Thermo Fisher Scientific (1 mentions)
Dab 3 3 diaminobenzidine, by Merck Group (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Trizol, by Merck Group (1 mentions)
Plasmid midiprep kit, by Qiagen (1 mentions)
Pgem t easy, by Thermo Fisher Scientific (1 mentions)
Calf intestinal alkaline phosphatase, by New England Biolabs (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Plasmid miniprep kit, by Merck Group (1 mentions)
Monolith nt 115, by NanoTemper (1 mentions)
17 protocols using anti his monoclonal antibody
1
Binding Analysis of MRP and Fibrinogen
2
Recombinant Protein Expression in E. coli and Insect Cells
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Strains used for the propagation of the plasmid DNA—E. coli DH5α, DH10BAC, for the expression of recombinant proteins cells—E. coli BL21 (DE3), insect cell line Sf21 and the cloning vectors pRSET-B, pFASTBAC-HTB, pGEM-3Z and pGEM–T Easy were procured from Invitrogen, USA. The restriction enzymes KpnI, NcoI and HindIII, Calf-Intestinal Alkaline Phosphatase, Pfu and Taq polymerases were obtained from New England Biolabs, USA. ATP and T7 RNA polymerase were purchased from Fermentas, USA. Nickel-NTA beads, DEAE-cellulose columns and plasmid midi-prep kit were procured from Qiagen, USA. PD-10 columns were purchased from GE Healthcare Life Sciences, USA. Trizol, DAB (3,3′-diaminobenzidine), Lysozyme, anti-His monoclonal antibody, plasmid miniprep kit and protease inhibitor cocktail were purchased from Sigma Chemicals, USA. Isopropyl β-d -1-thiogalactopyranoside (IPTG) was acquired from GIBCO-URL, USA. An antibody was raised in rabbit earlier against recombinant PPRV L protein domain 3 (1717–2183 a.a) expressed in E. coli [3 (link)]. γ-P32-ATP and α-P32-ATP were purchased from BRIT, Mumbai, India. The oligonucleotides were supplied by Sigma Chemical Co., India and were used to generate in vitro transcribed RNA substrate for RTPase assays.
3
SERBP1 Interaction with U3 Genes
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MST measurements were performed to test the direct interaction of SERBP1 with U3 genes using a NanoTemper Monolith NT.115 instrument (NanoTemper Technologies GmbH). SERBP1 protein was expressed in HEK293T cells by transfecting pcDNA3.1-SERBP1-HIS plasmid into the cells. Cells are lysed at 24h to collect protein for Western blotting using anti-HIS monoclonal antibody (Sigma, 1:6000) or labeled with RED-tris-NTA 2nd generation dye using the Monolith NT.115 Protein Labelling Kit for MST analysis. The U3 nucleic acid DNA fragments were amplified and purified using the oligonucleotides without biotin labeling. DNA-protein affinity interaction measurements were performed at 24°C using 40% LED power and 60% microscale thermophoresis power. All experiments were repeated twice for each measurement. Data were analyzed using NanoTemper analysis software.
4
Oral Delivery of scFv in Pigs
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Two crossbred (large white) pigs were oral administrated with 50 mL of purified scFv using a flexible gavage feeding needle (Tianyuan Animal instrument Inc., Shandong, China). At 4 h post inoculation, pigs were humanely euthanized with 100% CO2. The section of jejunum was collected, fixed in 10% formalin for 24 h and immersed in 30% sucrose for 48 h. Tissue samples were then embedded in OCT compound (Sakura Finetek, Radnor, PA, USA) and sectioned at 10 µm thickness using a cryomicrotome (Leica CM1900; Wetzlar, Germany). Cryosectioned samples were transferred onto the polylysine pre-treated slides, stained with anti-His monoclonal antibody (Sigma-Aldrich) as primary antibody followed by FITC conjugated goat anti-mouse antibody as secondary antibody. The sections were omitted primary antibodies as negative control. The samples were visualized by a fluorescence microscopy (Nikon).
5
Western Blot Analysis of E. coli Recombinant Proteins
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E. coli BL21(DE3) strains transformed with pBAD-PsCRN63, pBAD-PsCRN115, or pBAD empty vector were grown in LB broth at 37°C for several hours until the optical density (OD600) reached 0.3. Final concentrations of 0.1% and 2% L -arabinose were used to induce gene expression. After 5 h of induction, bacterial cells were collected and lysed for western blot. Samples were separated by standard SDS-PAGE gels and transferred to a PVDF membrane and then blocked with a PBST solution containing 5% non-fat milk for 30 min in room temperature with 60 rpm shaking. Anti-His monoclonal antibody (Sigma-Aldrich) and anti-β-RNAP (β-RNA polymerase) antibody (Abcam) were separately added to the PBST solution and incubated at 4°C overnight. Then the membranes were washed three times with PBST, followed by incubation with goat anti-mouse IRDye 800CW antibody (Odyssey) and goat anti-rabbit IRDye 800CW antibody (Odyssey) in PBST solution with 5% non-fat milk at room temperature for 45 min. The membranes were washed three times with PBST and visualized using a LI-COR Odyssey scanner with excitation at 700 and 800 nm.
6
Molecular Probing of Cellular Pathways
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LPA (15μM, Cat no. L7260, Sigma Aldrich), PDGF (60ng/μl; Cat no. P4306, Sigma Aldrich), Wortmannin (100nM, Cat no. W1628, Sigma Aldrich), anti-His monoclonal antibody (1:7000, Cat. no. MA1-21315, Invitrogen), G418 (6μg/ml, Cat no.1720, Sigma Aldrich), anti-HA monoclonal antibody (1:130 (IF), Cat no. sc-7392, Santa Cruz Biotechnology), anti-Cysteine synthase polyclonal antibody (1:1000 (IB), kind gift from Dr. Tomoyoshi Nozaki), anti-Hgl monoclonal antibody (1:50(IB;3F4-7F4) 1:130(IF; 3F4), kind gift from Dr. William Petri), anti-HA monoclonal antibody (1:1000 (IB), C29F4, Cell signaling technology), anti-GFP polyclonal antibody (1:500 (IB), Roche) Texas Red Dextran (100μg/ml, Cat. D-1864, Life Technologies), Alexa568 labelled Phalloidin (1:40; Cat No. A12380; Thermo Fisher Scientific), CellTracker Orange CMRA Dye (2 μM; C34551), Mowiol (Cat. 81381-250G, Sigma Aldrich), Alexa Fluor 680 anti-rabbit antibody (1:10,000 (IB); A-21076; Thermo Fisher Scientific), Alexa Fluor 800 anti-mouse antibody (1:10,000 (IB); A- 32730; Thermo Fisher Scientific)
7
Protein Purification and Characterization
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The eluted fractions were analyzed for yield and purity on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Protein samples were mixed with 2X SDS-PAGE loading buffer and boiled for 5 min at 100 °C; 10 µl of lysate, wash and eluted fractions were analyzed. Separated proteins were Coomassie blue stained and the fractions containing protein were pooled and dialyzed overnight against TEN10P buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 10 mM NaCl, 250 µM PMSF, 0.05% NP40).
Dialyzed protein was quantified by Qubit® 2.0 Fluorometer (Invitrogen, Paisley, UK) and stored in aliquots with 10% glycerol at -20 °C or analyzed by Western blotting. Separated proteins by SDS-PAGE were transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membranes were incubated with anti-His monoclonal antibody (Sigma, Missouri, USA) or anti-OPG polyclonal antibody (Abcam, Cambridge, UK).
After washing, horseradish peroxidase (HRP) conjugated anti-mouse or anti-rabbit IgG antibody (Jackson ImmunoResearch, Philadelphia, USA) was added. Immunoreactive protein bands were detected with the SuperSignal system (Pierce, Illinois, USA) according to the manufacturer's instructions.
Dialyzed protein was quantified by Qubit® 2.0 Fluorometer (Invitrogen, Paisley, UK) and stored in aliquots with 10% glycerol at -20 °C or analyzed by Western blotting. Separated proteins by SDS-PAGE were transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membranes were incubated with anti-His monoclonal antibody (Sigma, Missouri, USA) or anti-OPG polyclonal antibody (Abcam, Cambridge, UK).
After washing, horseradish peroxidase (HRP) conjugated anti-mouse or anti-rabbit IgG antibody (Jackson ImmunoResearch, Philadelphia, USA) was added. Immunoreactive protein bands were detected with the SuperSignal system (Pierce, Illinois, USA) according to the manufacturer's instructions.
8
Western Blot Protein Detection
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The separated proteins on an SDS-polyacrylamide gel were transferred onto a nitrocellulose membrane using a semi dry blotter (Trans-Blot SD, BioRad, USA). The nitrocellulose membrane was then blocked with skimmed milk [10% (w/v)] for 1 hour at room temperature. After three times of washing with TBS-T [50 mM Tris-HCl, pH 7.6; 150 mM NaCl; 0.1% (v/v) Tween 20], the nitrocellulose membrane was incubated with the anti-His monoclonal antibody (Merck, Germany) for 1 hour at room temperature. Afterwards, the nitrocellulose membrane was washed with TBS-T and incubated with the alkaline phosphatase conjugated goat anti-mouse antibody (Merck, Germany; 1:5000 dilutions) for 1 hour at room temperature. The immunoblotted bands were developed by adding 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) in alkaline phosphatase buffer [100 mM Tris-HCl; pH 9.5, 100 mM NaCl, 5 mM MgCl2]. Color development was terminated by incubating the nitrocellulose membrane in distilled water and the membrane was allowed to dry at room temperature.
9
GST-fused RIAM Protein Binding Assay
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Purified GST-fused RIAM or GST (10 µg each) was immobilized on 40 µL of glutathione-Sepharose 4B resin and equilibrated in the binding buffer consisting of 10 mM Tris, pH 7.5, 50 mM NaCl, 10% glycerol (v/v), 0.1% NP-40 (v/v), and Complete EDTA-free Protease Inhibitor (Roche, Indianapolis, IN). The desired His-tagged protein(s) were added in either GST-RIAM or GST immobilized binding mixture, and incubated at 4 °C for 2 hours. The beads were extensively washed for three times by the binding buffer. The bound proteins were eluted in 30 µL of 20 mM reduced glutathione (Sigma, St. Louis, MO) in the binding buffer and dissolved in 30 µL of 2X SDS sample buffer. The samples were resolved by 18% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Billerica, MA) for western blot analysis. The bound proteins were probed with either anti-His monoclonal antibody (120 ng ml−1, EMD Millipore, cat. No. 70796-4) or anti-GST monoclonal antibody (0.1 µg ml−1, Calbiochem, cat. No. OB03) and developed with Pierce ECL Western Blotting Substrate (Thermo Scientific, Portsmouth, NH). The binding experiments were performed in duplicate.
10
GST-fused RIAM Protein Binding Assay
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