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Product details for His-Tag antibody
His-Tag antibody
Overview
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| Antigen: | His-Tag |
| Antibody Type: | Monoclonal |
| Application: | Western Blotting (WB) |
| Host: | Mouse |
| Quantity: | |
| Price: | 293,65 $ (Plus shipping costs , $13.59 dry ice fee and VAT, Invoice in EUR) |
| Order number: | ABIN131923 |
| Availability: | 7 to 10 days until shipment |
Additional Information: His-Tag antibody
| Antigen | His-Tag |
| Immunogen | This antibody was purified from hybridoma (clone 6C4) supernatant using protein A agarose. This hybridoma was established by fusion of mouse myeloma cell P3-U1 with Balb/c mouse splenocyte immunized with synthetic peptide 6 His. |
| Antibody Type | Monoclonal |
| Isotype | IgG1 »Matching secondary antibodies |
| Description | A variety of plasmids contain DNA that encodes an N-terminal tag consisting of six histidine (His) residues, followed by an extended multiple cloning sites. The His-Tag fusion protein expression system is commonly used because the 6 x His tag on the recombinant proteins allows for efficient coupling to Ni ++ affinity resins and purification by a single step chromatography. This specific antibody for His-Tag fusion protein is useful for monitoring of the fusion protein expression and affinity purification. |
| Clone | 6C4 |
| Host | Mouse |
| Specificity | This antibody reacts with His-Tag on Western blotting. |
Application Details: His-Tag antibody
| Application | Western Blotting (WB) |
| Application Notes | SDS-PAGE & Western Blotting 1) Wash the cells 3 times with PBS and suspend with 10 volumes of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol) containing appropriate protease inhibitors. Incubate it at 4 o C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the Lysis buffer to make 8 mg/mL solution. 3) Mix the sample with equal volume of Laemmli’s sample buffer. 4) Boil the samples for 2 minutes and centrifuge. Load 10 µL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system (Transfer Buffer - 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for precise transfer procedure. 6) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4 o C. 7) Incubate the membrane with primary antibody diluted with PBS, pH7.2 containing 1% skimmed milk as suggest in the Applications for 1 hour at room temperature. (The optimal antibody concentration will depend on the experimental conditions.) 8) Wash the membrane with PBS (5 minutes x 6 times). 9) Incubate the membrane with the 1:10,000 HRP-conjugated anti-mouse IgG (MBL, code no. 330) diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 10) Wash the membrane with PBS (5 minutes x 6 times). 11) Wipe excess buffer from the membrane, then incubate it with appropriate chemiluminescence reagents for 1 minute. Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap. 12) Expose to X-ray film in a dark room for 10 minutes. Develop the film as usual. The conditions for exposure and development may vary. 050117-1 Western blotting: 1 µg / mL. Immunoprecipitation: Not tested. Immunohistochemistry: Not tested. Immunocytochemistry: Not tested. Flow cytometry: Not tested. |
| Concentration | 1 mg/mL |
| Buffer | 100 µg IgG in 100 µL P BS containing 50% glycerol, pH 7.2. Contains no preservatives. |
| Research Area | Tags/Labels |
| Restrictions | For Research Use Only |
Publications: His-Tag antibody
| Publications |
Porath: "Immobilized metal ion affinity chromatography." In: Protein expression and purification , Vol. 3, Issue 4, pp. 263-81, 1992/01 (PubMed) |
Images: His-Tag antibody
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Western blot analysis of His-Azami-Green (1) and His-EGFP (2) using ABIN131923. |
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