FZR1 Antikörper

Produktnummer ABIN487309

Dieses Maus Monoklonal-Antikörper erkennt spezifisch FZR1 in WB und IP. Er zeigt eine Reaktivität gegenüber Human, Maus und Ratte.

Kurzübersicht für FZR1 Antikörper (ABIN487309)

Target: FZR1 (Fizzy/cell Division Cycle 20 Related 1 (FZR1))

Reaktivität: Human, Maus, Ratte

Wirt: Maus

Klonalität: Monoklonal

Konjugat: Dieser FZR1 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB), Immunoprecipitation (IP)

Klon: DCS-266

Produktdetails anti-FZR1 Antikörper

Kreuzreaktivität (Details): Species reactivity (tested):Human, Mouse and Rat.

Produktmerkmale: Synonyms: Cdh1/Hct1 homolog, CDC20-like protein 1, Fizzy-related protein homolog, FYR, FZR,KIAA1242

Aufreinigung: Protein-A Sepharose Chromatography.

Immunogen: Recombinant Human CDH1 protein. Remarks: Hybridoma was established by fusion of Mouse myeloma cell NS-2 with Balb/cmouse splenocyte

Isotyp: IgG1

Anwendungsinformationen

Applikationshinweise: Western Blot: 1-5 μg/mL for chemiluminescence detection system. Positive Controls: Jurkat, NIH/3T3, C2C12 and Rat-1 cells. Immunoprecipitation: 1-2 μg/200-500 μL of cell extract from 5 x 10^6 cells. . Positive Control: HeLa Cells.
Other applications not tested.
Optimal dilutions are dependent on conditions and should be determined by the user. Species Reactivity: Tested: Human, Mouse and Rat. Add. Information: This product was originally produced by MBL International.

Protokoll: SDS-PAGE & Western Blotting1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mMTris-HCl, pH 7. 2, 250 mM NaCl, 0. 1% NP-40, 2 mM EDTA, 10% glycerol) containingappropriate protease inhibitors. Incubate it at 4°C with rotating for 30 minutes, thensonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C and transfer the supernatant toanother tube. Measure the protein concentration of the supernatant and add the Lysisbuffer 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 1mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hourin a semi-dry transfer system. (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for the transfer procedure. 6) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH7. 2) for 1 hour at room temperature, or overnight at 4°C. 7) Incubate the membrane with primary antibody diluted with PBS, pH 7. 2 containing 1%skimmed milk as suggested in the APPLICATIONS for 1 hour at room temperature. (Theconcentration of antibody will depend on the conditions. )8) Wash the membrane with PBS (5 minutes x 6 times). 9) Incubate the membrane with the 1: 10000 POD-conjugated anti-mouse IgG diluted with1% 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 appropriatechemiluminescence reagents for 1 minute. Remove extra reagent from the membrane bydabbing with a paper towel, and seal it in plastic wrap. 12) Remove extra reagent from the membrane by dabbing with paper towel, and seal it inplastic wrap. 13) Expose to an X-ray film in a dark room for 3 minutes. 14) Develop the filmas usual. The condition for exposure and development may vary. Positive Controls for Western blotting: Jurkat, NIH/3T3, C2C12, Rat-1Immunoprecipitation1) Collect the cultured cells from 75-cm2 flask (containing about 0. 5-1 x 10e7 cells). 2) Wash the cells 2 times with PBS and suspend with 400 µL of cold Lysis buffer (50 mMHEPES-KOH, pH 7. 5, 250 mM NaCl, 0. 1% NP-40, 5 mM EDTA,10% glycerol) containingappropriate protease inhibitors. Incubate it at 4°C with rotating for 30 minutes, thensonicate briefly (up to 10 seconds). 3) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C and transfer the supernatant toanother tube. 4) Add 50 µL of 50% protein G agarose beads in the supernatant. Incubate it at 4°C withrotating for 60 minutes. 5) Centrifuge the tube at 12,000 x g for 5 minutes at 4°C. Supernatant is equally dividedinto another two tube. 6) Add the mouse IgG1 isotype control antibody or anti-Cdh1/Fzr (DCS-266) monoclonalantibody at the amount as suggested in the APPLICATIONS to the supernatant. Vortexbriefly and incubate with gently agitation for 30-120 minutes at 4°C. 7) Add 20 µL of 50% protein G agarose beads into the tube. Mix well and incubate withgentle agitation for 30-60 minutes at 4°C.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Konzentration: 1.0 mg/mL

Buffer: PBS, pH 7.2 containing 50 % Glycerol without preservatives.

Konservierungsmittel: Without preservative

Lagerung: -20 °C

Informationen zur Lagerung: Store the antibody undiluted at -20 °C.
Shelf life: one year from despatch.

Haltbarkeit: 12 months

Details zu FZR1

Target: FZR1 (Fizzy/cell Division Cycle 20 Related 1 (FZR1))

Andere Bezeichnung: FZR1 / CDH1

Hintergrund: Two highly conserved WD40-repeat proteins, Cdc20 and Cdh1/Hct1, have been identified as limiting, substrate-specific regulators for anaphase-promoting complex (APC)-dependent proteolysis. Cdc20 and Cdh1 bind directly to APC and activate its cyclin ubiquitination activity. Cdc20 activates APC at the onset of anaphase, while Cdh1 activates APC from late anaphase through G1. Phosphorylation of Cdh1 by cyclin dependent kinases (Cdks), such as Cdc28, blocked Cdh1-APC interaction and APC activation, while dephosphorylation by the phosphatase Cdc14 restored this activity. Activation of the APC and cyclin degradation is required for exit from mitosis, hence, the phosphorylation of Cdc20 and Cdh1 by Cdks regulate APC activity and may control the precise progression of mitosis.Synonyms: CDC20-like protein 1, Cdh1/Hct1 homolog, FYR, FZR, Fizzy-related protein homolog, KIAA1242

Gen-ID: 51343

UniProt: Q9UM11

Pathways: DNA Replication, Synthesis of DNA