Azami-Green Antikörper

Antigen: Azami-Green

Klonalität: Monoklonal (3D10)

Wirt: Maus

Applikation: Immunpräzipitation (IP)

Produktnummer: ABIN131944

Produktbeschreibung

Immunogen: This antibody was purified from hybridoma (clone 3D10) supernatant using protein A column. This hybridoma was established by fusion of mouse myeloma cell P3-U1 with C3H mouse lymphocyte immunized with recombinant Azami-Green.

Isotyp: IgG2a  (Passende Sekundärantikörper)

Klon: 3D10

Beschreibung: CoralHue™ Azami Green (AG) was cloned from the stony coral whose Japanese name is “Azami-Sango. It absorbs light maximally at 492 nm and emits green light at 505 nm. Wild-type AG rapidly matures to form a tetrameric complex. AG has been carefully engineered to form a monomer, CoralHue™ Monomeric Azami-Green (mAG1) that maintains the brightness and pH stability of the parent protein. AG and mAG1 can be used to mark cells, label proteins or subcellular structures, or to report gene expression without problems stemming from protein aggregation.

Spezifität: This antibody clearly immunoprecipitates both of tetramer and monomer form of CoralHue™ Azami-Green. The antibody also shows some cross-reactivity with CoralHue™ Kaede and very slight cross-reactivity with CoralHue™ Midoriishi-Cyan and CoralHue™ Dronpa in immunoprecipitation experiments.

Anwendungen

Applikationshinweise: Immunoprecipitation 1) Add primary antibody as suggested in the Applications into 30 µL of 50% protein A agarose beads resuspended in the cold Lysis buffer (50 mM Tris-HCl pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol). Mix well and incubate with gentle agitation for 60 minutes at 4 o C. 2) Wash the beads 3-5 times with the cold Lysis buffer Im m unoprecipitation of A zam i-G reen from E.coli with n o rm al m ouse IgG 2a (1) or M 103-3 (2). After im m unoprecipita te d w ith th e a n tibody, immunocomplex w as resolved on SD S-PA G E and immunoblotted with P M 002 (A nti-H is-Tag polyclonal a n tibody). IgG Heavy Chain Azami-Green IgG Light Chain 12 kD a 66 20 30 45 Im m unoprecipitation of A zam i-G reen from E.coli with n o rm al m ouse IgG 2a (1) or M 103-3 (2). After im m unoprecipita te d w ith th e a n tibody, immunocomplex w as resolved on SD S-PA G E and immunoblotted with P M 002 (A nti-H is-Tag polyclonal a n tibody). IgG Heavy Chain Azami-Green IgG Light Chain 12 kD a 66 20 30 45 IgG Heavy Chain IgG Heavy Chain Azami-Green Azami-Green IgG Light Chain IgG Light Chain 12 kD a 66 20 30 45 12 kD a 66 20 30 45 kD a 66 20 30 45 (centrifuge the tube at 2,500 x g for 10 seconds). 3) Add 100 µL of the recombinant protein. Mix well and incubate with gentle agitation for 60 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 beads in 20 µL of Laemmli’s sample buffer and 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. 6) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 10V for 45 minutes 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. 7) To reduce nonspecific binding, soak the membrane in 100% Block Ace for 1 hour at room temperature, or overnight at 4 o C. 8) Incubate the membrane with the 1:1,000 anti-His-Tag antibody (MBL, code no.PM002) diluted with 10% Block Ace (in PBS, pH 7.2) for 1 hour at room temperature. 9) Wash the membrane with PBS (5 minutes x 6 times). 10) Incubate the membrane with the 1:10,000 HRP-conjugated anti-rabbit IgG (BioRad, code no. 170-6515) diluted with 1% BSA (in PBS, pH 7.2) for 1 hour at room temperature. 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 an X-ray film in a dark room for 2 minutes. Develop the film as usual. The conditions for exposure and development may vary. 050609-1 Western blotting: Not recommended. Immunoprecipitation: 2 µg / 100 µL of the recombinant protein. Immunohistochemistry: Not tested. Immunocytochemistry: Not tested. Flow Cytometry: Not tested.

Konzentration: 1 mg/mL

Buffer: 100 µg IgG in 100 µL PBS containing 50% glycerol, pH 7.2. Contains no preservatives.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Bilder

Immunoprecipitation of A zam i-G reen from E.coli w ith norm al m ouse IgG 2a (1) or ABIN131944 (2) . A fter immunoprecipitated with the antibody, immunocomplex was resolved on S D S -P A G E and im m unoblotted w ith PM002 (A nti-H is-T ag polyclonal antibody) .

Publikationen

Publikationen: Karasawa, Araki, Yamamoto-Hino et al.: "A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling." in: The Journal of biological chemistry, Vol. 278, Issue 36, pp. 34167-71, 2003 (PubMed).

Karasawa, Araki, Nagai et al.: "Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer." in: The Biochemical journal, Vol. 381, Issue Pt 1, pp. 307-12, 2004 (PubMed).