Kaede Antikörper

Antigen: Kaede

Klonalität: Monoklonal (2F4)

Wirt: Maus

Applikation: Immunpräzipitation (IP)

Produktnummer: ABIN131950

Produktbeschreibung

Immunogen: This antibody was purified from hybridoma (clone 2F4) supernatant using protein A column. This hybridoma was established by fusion of mouse myeloma cell P3-U1 with C3H mouse lymphocyte immunized with recombinant Kaede.

Isotyp: IgG1  (Passende Sekundärantikörper)

Klon: 2F4

Beschreibung: CoralHue™ Kaede protein emits green fluorescence that can be irreversibly converted to red. The red fluorescence is comparable in intensity to the green and is stable under usual aerobic conditions. The green-to-red conversion is highly sensitive to irradiation with UV or violet light (350-410 nm). Maximal illumination results in a 2,000-fold increase in the ratio of red-to-green signal. The excitation lights used to elicit red and green fluorescence do not induce the photoconversion. CoralHue™ Kaede provides a simple and powerful technique for regional optical marking.

Spezifität: This antibody clearly immuno- precipitates CoralHue™ Kaede. The antibody also exhibits slight cross-reactivity with CoralHue™ Midoriishi-Cyan and CoralHue™ Dronpa.

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 (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). Im m unoprecipita tio n o f K a e d e fro m E.coli w ith norm al m ouse IgG1 (1) or M 106-3 (2). After im m unoprecipitate d w ith th e a n tibody, im m unocom plex w as resolved on SDS-PA GE and im m unoblotted with P M 0 0 2 (A n ti-His-Tag polyclonal a n tibody). IgG Heavy chain Kaede IgG Light Chain 66 45 30 20 12 kD a Im m unoprecipita tio n o f K a e d e fro m E.coli w ith norm al m ouse IgG1 (1) or M 106-3 (2). After im m unoprecipitate d w ith th e a n tibody, im m unocom plex w as resolved on SDS-PA GE and im m unoblotted with P M 0 0 2 (A n ti-His-Tag polyclonal a n tibody). IgG Heavy chain Kaede IgG Light Chain 66 45 30 20 12 kD a IgG Heavy chain IgG Heavy chain Kaede Kaede IgG Light Chain IgG Light Chain 66 45 30 20 12 kD a 66 45 30 20 12 kD a 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 Kaede from E.coli w ith norm al m ouse IgG 1 (1) or ABIN131950 (2) . A fter im m unoprecipitated w ith the antibody, im m unocom plex w as 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: Ando, Hama, Yamamoto-Hino et al.: "An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, Issue 20, pp. 12651-6, 2002 (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).