HA-Tag-Agarose Antikörper

Antigen: HA-Tag-Agarose

Klonalität: Polyklonal

Wirt: Kaninchen

Applikation: Immunpräzipitation (IP)

Produktnummer: ABIN130393

Produktbeschreibung

Immunogen: This antibody was purified from rabbit serum using protein A agarose. The rabbit was immunized with carrier protein (CP) conjugated synthetic peptide, CP-YPYDVPDYA (HA-Tag).

Beschreibung: Epitope tagging is a powerful and versatile strategy for detecting and purifying proteins expressed by cloned genes. Short sequences encoding the epitope tag are cloned in-frame with target DNA to produce fusion proteins containing the epitope tag peptide. Due to their small size, epitope tags do not affect the tagged protein’s biochemical properties. Anti-epitope tag antibodies can serve as universal purification or detection reagents for any tag-containing protein. With this technique, the gene expression can be easily monitored by Western blotting, immunoprecipitation and immunofluorescence. Amino acid sequences that are commonly used for epitope tagging include peptide sequences corresponding to portions of Influenza hemagglutinin protein, human c-myc gene product and Vesicular stomatitis virus glycoprotein.

Spezifität: This antibody recognizes the HA-Tag peptide sequence (YPYDVPDYA) and is suitable for immunoprecipitation assays.

Anwendungen

Applikationshinweise: Immunoprecipitation 1) Wash the cells 3 times with PBS and suspend with 10 volumes of cold Lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% NP-40) 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. 3) Add agarose as suggested in the Applications into 200 µL of cell extract. Mix well and incubate with gentle agitation for 30-120 minutes at 4 o C. 4) Wash the beads 3-5 times with the cold Lysis buffer (centrifuge the tube at 2,500 x g for 10 seconds). 5) Resuspend the agarose in 20 µL of Laemmli’s sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. 6) Load 10 µL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 7) 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. 8) To reduce nonspecific binding, soak the membrane in 5% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4 o C. 9) Incubate the membrane with 1:1,000 diluted anti-HA-Tag polyclonal antibody (MBL, code no. 561) diluted with PBS, pH 7.2 containing 1% skimmed for 1 hour at room temperature. (The optimal antibody concentration will depend on the experimental conditions.) 10) Wash the membrane with PBS (5 minutes x 3 times). 11) Incubate the membrane with the 1:10,000 HRP-conjugated anti-mouse IgG (MBL, code no. 458) diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 12) Wash the membrane with PBS (5 minutes x 3 times). 13) Wipe excess buffer from the membrane, then incubate it with appropriate chemiluminescence reagents for 1 min- ute. Remove extra reagent from the membrane by dab- bing with a paper towel, and seal it in plastic wrap. 14) Expose to an X-ray film in a dark room for 3 minutes. Develop the film as usual. The conditions for exposure and development may vary. (Positive control for Immunoprecipitation: transfectant) 060920-1 kDa 1 2 50 - 30 - 35 - 25 - IgG heavy chain HA-tagged protein kDa 1 2 50 - 30 - 35 - 25 - 50 - 50 - 30 - 30 - 35 - 35 - 25 - 25 - IgG heavy chain HA-tagged protein IgG heavy chain HA-tagged protein Immunoprecipitation of HA-Tag from 293T/pcDNA-HA-tagged protein cell lysate with rabbit IgG (1) and anti-HA- Tag-Agarose (2). After immunoprecipitated with the antibody, immunocomplex was resolved on SDS-PAGE and immunoblotted with 561. Western blotting: Not tested. Immunoprecipitation: 20 µL / 200 µL of cell extract from 5x10 6 cells

Buffer: 540 µg of anti-HA Tag polyclonal antibody covalently coupled to 200 µL of agarose gel and provided as a 50% gel slurry suspended in PBS containing preservative (0.09% sodium azide) for a total volume of 400 µL. * Azide may react with copper or lead in plu

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Bilder

Immunoprecipitation of HA-Tag from 293T/pcDNA-HA-tagged protein cell lysate with rabbit IgG (1) and anti-HATag-Agarose (2). After immunoprecipitated with the antibody, immunocomplex was resolved on SDS-PAGE and immunoblotted with ABIN130391.

Publikationen

Publikationen: Field, Nikawa, Broek et al.: "Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method." in: Molecular and cellular biology, Vol. 8, Issue 5, pp. 2159-65, 1988 (PubMed).

Heald, McLoughlin, McKeon: "Human wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase." in: Cell, Vol. 74, Issue 3, pp. 463-74, 1993 (PubMed).