After the electrophoresis, proteins separated on the gel were transferred onto a nitrocellulose membrane (pore size 0.2 µm, Bio-Rad, Hercules, CA, USA) for 120 min using the Trans-blot SD semi-dry electrophoretic transfer cell (Bio-Rad) at 70 V (voltage constant). The membrane was blocked for 1 h at room temperature with 5% skim milk (Wako Pure Chemical Industries) in Phosphate buffered saline containing 0.1% Tween-20 (PBS-T). The membrane was then incubated for 8 h at 4 °C with a primary antibody, mouse anti-His tag antibody (Cell Signaling Technologies, Danvers, MA, USA), at 1:1000 dilution in PBS-T containing 5% skim milk. After washing three times with PBS-T for 15 min, the membrane was incubated for 1 h at room temperature with a secondary antibody, HRP-conjugated anti-mouse IgG antibody (Cell Signaling Technologies), diluted by 1:1000 ratio in PBS-T containing 5% skim milk. The membrane was washed three times with PBS-T for 15 min and then treated with Western Blot detection solution A and B of Pico EPD Kit (ELPIS Biotech, Daejeon, South Korea) by 1:1 ratio for visualizing protein bands. Photographs of blots were taken by the Chemidoc XRS+ illumination system (Bio-Rad).
Mouse anti his tag antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Mouse anti-His tag antibody is a monoclonal antibody that specifically recognizes the polyhistidine (His) tag, a commonly used protein tag for recombinant protein expression and purification. This antibody can be used to detect and identify His-tagged proteins in various applications such as Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Skim milk, by Cell Signaling Technology (1 mentions)
Skim milk, by Fujifilm (1 mentions)
Hrp conjugated anti mouse igg antibody, by Cell Signaling Technology (1 mentions)
Trans blot sd semi dry electrophoretic transfer cell, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Mn2 phos tag, by Fujifilm (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Oligonucleotide, by Merck Group (1 mentions)
Mouse anti gfp, by Santa Cruz Biotechnology (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Peroxidase conjugated goat anti mouse igg, by Beyotime (1 mentions)
Ni nta column, by GE Healthcare (1 mentions)
6 protocols using mouse anti his tag antibody
1
Western Blot Protein Detection Protocol
2
Kinase Activity Assay Protocol
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Ptk1 (5 μg) was incubated with 5 μg Php1 in a total volume of 50 μl containing 100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM DTT, and 1 mM MnCl2 at 37°C for 2 h unless otherwise indicated. The reactions were stopped by adding 5× sample buffer and boiling for 10 min. Samples were separated by SDS-PAGE or Mn2+-Phos-tag (Wako)–SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked (TBS containing 0.1% Tween 20 [TBST] and 5% bovine serum albumin) at room temperature for 1 h and washed with TBST. Membranes were probed with mouse antiphosphotyrosine antibody (clone PY20; 1:1,000; Sigma), mouse anti-His-tag antibody (Cell Signaling), or rabbit anti-GST-tag antibody (Cell Signaling) at 4°C overnight. After washing (3× TBST), membranes were incubated with a horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibody (1:1,000; Cell Signaling) at room temperature for 1 h. Immunoreactive bands were detected with Pierce ECL Western blotting substrate (Thermo Fisher) and a ChemiDoc XRS+ imaging system (Bio-Rad).
3
Glycan Array Analysis and Quantification
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Glycans were prepared for printing as described by Day et al. (34 (link)). Glycan array slides were printed SuperEpoxy 3-activated substrates using the glycan library previously described by Arndt et al. (35 (link)) and Day et al. (25 (link)). Table S1 in the supplemental material gives a full list of the structures printed. The glycan arrays were performed and analyzed as previously described (27 (link)). Briefly, 2 μg of protein in phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) containing 1 mM MgCl2 and 1 mM CaCl2 was precomplexed with a mouse anti-His tag antibody (Cell signaling) and two Alexa 488-labeled antibodies (rabbit anti-mouse and goat anti-rabbit) at a molar ratio of 1:1:0.5:0.25 to enable detection. Protein was incubated on the slide for 30 min and washed three times in PBS. Slides were scanned on a PerkinElmer ProScan four-laser scanner and analyzed using ScanArray Express and Microsoft Excel.
4
GFP Antibody Characterization Protocol
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Oligonucleotides were purchased from Sigma‒Aldrich (Bangalore, India). Mouse anti-GFP was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and mouse anti-His tag antibodies were purchased from Cell Signaling Technology (Denvers, MA). Restriction enzymes, NEB Hi-Fi builder (E2621L) and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA). DNA polymerases were purchased from GeNei (Bangalore, India) and Thermo Fisher Scientific (Waltham, MA).
5
Recombinant EbSWPs Characterization by SDS-PAGE
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In SDS-PAGE analysis of recombinant EbSWPs, 40 μL protein was added into 10 μL 5×SDS loading buffer and incubated in a water bath at 100°C for 10 min. After polyacrylamide gel electrophoresis of the mixture, the protein in the gel was transferred onto a polyvinylidene fluoride membrane (Millipore). The membrane was blocked with 5% skimmed milk powder for 2 h and reacted with mouse anti-His-tag antibodies (Cell Signaling Technology). Peroxidase-conjugated goat anti-mouse IgG (Beyotime Technology, Shanghai, China) was used as the secondary antibody.
6
Cloning and Purification of Mycoplasmal TrmFO
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M. bovis HB0801 chromosomal DNA was used as template for amplification of the trmFO gene. TGA is a universal termination codon but it encodes tryptophan in mycoplasmas. When cloning a mycoplasmal gene in the E. coli expression system, the presence of TGA codon can lead to the early termination of gene translation. In this study, the TGA codon is not found in the coding sequence of the trmFO gene. Specific primers for amplifying trmFO were designed with Oligo 7 software (Molecular Biology Insights, West Cascade, CO, USA) as follows: F5′-GGCGGTACC ATGAAAAAAATAAGAGTTATTG-3′ and R5′-CGGGGATCC CTATAAATTTTGCTTAATAAAC-3′ (underlined sequences represent KpnI and BamHI restriction sites, respectively). The PCR product was cloned into pET-30a (+) vector (Novagen, Madison, WI, USA). His-tagged TrmFO was expressed in E. coli BL21 and purified by Ni-NTA columns affinity chromatography under native conditions according to the manufacturer’s instructions (GE Healthcare, Boston, MA, USA). The purified rTrmFO was analyzed by SDS-PAGE and by western blotting using mouse anti-His tag antibodies (Cell Signaling Technology, Danvers, MA, USA). Protein concentration was quantified using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).
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