First, 96-well plates were washed with H2O and then incubated overnight with 0.1 mg ml−1 of calf thymus histones (Sigma) at 4 °C. Next, the plates were blocked with 5% BSA in PBST (PBS containing 0.1% Tween-20) for 30 min. The absorbed histones were mock ADP-ribosylated in the absence of NAD+ or ADP-ribosylated in the presence of 50 μM NAD+ (Sigma) in PARP1 reaction buffer (50 mM Tris–HCl pH 8, 0.8 mM MgCl2, 1% glycerol and 1.5 mM DTT) containing 40 nM single-stranded oligodeoxyribonucleotide (5′-CATATGCCGGAGATCCGCCTCC-3′) and 10 nM high-specific-activity PARP1 (Trevigen) for 2 h. After subsequent washes with PBST, 500 nM of XRCC1-His, XRCC1-HisRK, His–XRCC1161–406, His–APLF, His–APLFZFD and His–USP3 were added and incubated for 30 min on ice in Dilution buffer (20 mM Tris–HCl pH 7.5 and 130 mM NaCl). The plates were washed with PBST and incubated for 30 min with Dilution buffer containing mouse anti-polyhistidine antibody (Sigma) followed by three washes with PBST and subsequent incubation with horseradish peroxidase-conjugated secondary antibody (DAKO) for 30 min. After washing out the secondary antibody, 3,3′,5,5′-tetramethylbenzidine liquid substrate (slow kinetic form; Sigma) was added to the wells for 5 min. The reactions were stopped by the addition of 0.2 M HCl. Absorbance was read at 450 nm using a CLARIOstar microplate reader (BMG Labtech).
Mouse anti polyhistidine antibody
Manufactured by Merck Group
Sourced in United States
The Mouse anti-polyhistidine antibody is a laboratory reagent used to detect and purify proteins with a polyhistidine tag. It is a monoclonal antibody that specifically binds to the polyhistidine sequence, allowing for the identification and isolation of tagged proteins in various applications.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Calf thymus histone, by Merck Group (1 mentions)
Parp1, by Merck Group (1 mentions)
Parp1, by Bio-Techne (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Clariostar microplate reader, by BMG Labtech (1 mentions)
Lsm510 meta nlo, by Zeiss (1 mentions)
Mouse anti vinculin antibody, by Merck Group (1 mentions)
Anti rabbit igg hrp conjugate antibody, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit trueblot igg hrp conjugate antibody, by Thermo Fisher Scientific (1 mentions)
Rabbit anti eif4e antibody, by Cell Signaling Technology (1 mentions)
Re blot plus mild solution, by Merck Group (1 mentions)
Image lab v5, by Bio-Rad (1 mentions)
5 protocols using mouse anti polyhistidine antibody
1
Histone ADP-ribosylation and XRCC1 Binding
2
Visualizing Intracellular Tat-PGAM1 Delivery
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The intracellular delivery of Tat-PGAM1 and Control-PGAM1 was visualized using immunocytochemical staining for polyhistidine. Briefly, coverslip-grown NSC34 cells were exposed to 3 μM Tat-PGAM1 or Control-PGAM1 protein for 60 min and the cells were fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.4) for 5 min at 25 °C. Thereafter, the cells were sequentially incubated with mouse anti-polyhistidine antibody (Sigma, St. Louis, MO, USA) and Alexa Fluor® 488-conjugated anti-mouse Immunoglobulin G (1:1000; Jackson ImmunoResearch, West Grove, PA, USA) with 1 μg/mL 4,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Waltham, MA, USA). The immunoreaction was observed under a confocal fluorescence microscope (LSM 510 META NLO; Zeiss GmbH, Jena, Germany) and the fluorescence was measured using a Fluoroskan ELISA plate reader (Labsystems Oy, Helsinki, Finland) at 485 nm excitation and 538 nm emission.
3
Far-Western Blot Analysis of Recombinant Protein Binding
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A far-Western blotting technique was employed to further characterize the specific binding interactions between the recombinant proteins and bovine fibrinogen (86 (link)). To dissociate native bovine fibrinogen into its constituent polypeptide chains (Aα, Bβ, and γ), 60 μl of bovine fibrinogen stock solution (1 mg/ml) was mixed with 350 μl of gel loading buffer (100 mM Tris-Cl [pH 6.8], 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM dithiothreitol), heated at 95°C for 5 min, and separated in Tris-glycine polyacrylamide gels by SDS-PAGE (4 to 20% gradient gel) at a constant voltage of 180 V for 50 min. The fibrinogen chains were electroblotted onto a nitrocellulose membrane (100 V, 240 mA, and 120 min), and the membrane was blocked with 5% (wt/vol) skimmed milk. Membranes were subsequently incubated with 30 μg/ml of recombinant protein. Any bound protein was detected by incubation of the membrane with mouse anti-polyhistidine antibody (Sigma-Aldrich, Dorset, UK), diluted 1:2,000, followed by goat anti-mouse antibody (Sigma-Aldrich) and development in 3,3′-diaminobenzidine membrane substrate (Sigma-Aldrich).
4
Optimized Western Blot Protocol
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Western blot (WB) was performed with 1/3000 mouse anti-polyHistidine antibody (Sigma-Aldrich, USA), 1/1000 rabbit anti-eIF4E antibody (Cell Signaling, Ozyme), 1/2500 anti-mouse IgG HRP conjugate antibody (Promega, France), 1/1000 anti-rabbit Trueblot IgG HRP conjugate antibody and 1/1000 anti-mouse Trueblot IgG HRP conjugate antibody (eBiosciences), 1/5000 anti-rabbit IgG HRP conjugate antibody (Santa Cruz Biotechnology, Germany), 1/5000 rabbit Hsp27 antibody (Assay Designs, FranceLoading levels were normalized using 1/2000 mouse anti-vinculin antibody (Sigma-Aldrich). Re-blot Plus Mild Solution (Millipore, France) was used for membrane stripping during 9 min at RT.
5
Quantitative Protein Analysis via SDS-PAGE and Western Blot
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Infiltrated leaves (7 DPI) were harvested and ground in liquid nitrogen to a fine powder. We then extracted 60 mg of leaf powder in 200 μl buffer K (62.5 mM Tris, pH 7.4, 10% glycine, 5% 2-mercaptoethanol, 2% SDS, 8 M urea). Ten microliter of the extract were mixed with loading buffer and boiled for 10 min before loading. Samples collected from the density gradient and IMAC procedures were mixed with 5x loading buffer, boiled at 100°C for 10 min and separated by reducing SDS-PAGE (12% polyacrylamide gel, 200 V for 90 min). Gels were stained with Coomassie Blue or transferred to a nitrocellulose membrane. The membrane was blocked with 5% (w/v) skimmed milk in phosphate buffered saline (PBS) for 1 h and then incubated with a mouse anti-poly-histidine antibody (Sigma-Aldrich Chemie GmbH, Germany) at room temperature for 2 h, diluted 1:10000. The blot was washed three times in PBS plus 0.05% Tween-20 (PBST) and then incubated for 1 h with the secondary anti-mouse alkaline phosphatase-conjugated antibody (diluted 1:5000). The membrane was washed another three times with PBST and the signal was detected using the NBT/BCIP system. For quantitation, the samples were compared to serial dilutions of a His6-tagged standard protein, and the images were analyzed using BioRad Image Lab v5.1.
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