219440 PhosphoDetect™ Anti-Cdk1 (pTyr¹⁵) Rabbit pAb

219440
  
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      Áttekintés

      Replacement Information

      Kulcsspecifikációk táblázata

      Host
      Rb
      Description
      Overview

      This product has been discontinued.





      Recognizes the ~34 kDa Cdk1 protein, phosphorylated at Tyr15, in hydroxyurea treated SaoS cell extracts. Also recognizes phosphorylated Cdk2. Does not react with Cdk4, Cdk6, Cdk7, or other phosphotyrosine proteins.
      Catalogue Number219440
      Brand Family Calbiochem®
      Application Data
      Detection of human Cdk1 phosphorylated at Tyr15 by immunoblotting. Samples: Whole cell lysate from Saos cells left untreated (left lane) or treated with hydroxyurea (middle lane) or nocodazole (right lane). Primary antibody: PhosphoDetect™ Anti-Cdk1 (pTyr15) Rabbit pAb (Cat. No. 219440) (1:1000). Detection: chemiluminescence.
      References
      ReferencesNorbury, C., and Nurse, P. 1992. Annu. Rev. Biochem. 61, 441.
      Hunter, T. 1995. Cell 80, 225.
      Galaktionov, K., et al. 1995. Genes Dev. 9, 1046.
      Watanabe, K., et al. 1995. EMBO J. 14, 1878.
      Atherton-Fessler, S., et al. 1993. Mol. Cell. Biol. 13, 1675.
      McGwan, C.H. and Russel, P. 1993. EMBO J. 12, 75.
      Product Information
      FormLiquid
      FormulationIn 150 mM NaCl, 10 mM sodium HEPES, 100 mg/ml BSA, 50% glycerol, pH 7.5.
      Positive controlSaos cells treated with hydroxyurea
      PreservativeNone
      Applications
      Key Applications Immunoblotting (Western Blotting)
      Immunocytochemistry
      Immunoprecipitation
      Application NotesImmunoblotting (1:1000)
      Immunoprecipitation (1:100)
      Immunocytochemistry (1:100)
      Application CommentsAlso recognizes phosphorylated Cdk2. Does not cross-react with Cdk4, Cdk6, Cdk7, or other phosphotyrosine proteins.

      Recommended Protocol for Immunoblotting

      Solutions and Reagents
      • Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol, pH 8.5
      • SDS Sample Buffer: 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromophenol blue.
      • 10X TBS (Tris-buffered saline): To prepare 1 liter, 24.2 g Tris base, 80 g NaCl, adjust pH to 7.6 with HCl. Dilute 1:10 for use.
      • Blocking Buffer: 1X TBS, 0.1% Tween®-20 detergent with 5% non-fat dry milk.
      • Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween®-20 detergent with 5% BSA
      • Wash Buffer (TBST): 1X TBS, 0,1% Tween®-20 detergent

      Blotting Membrane
      Nitrocellulose or PVDF membranes may be used.

      SDS-PAGE and Protein Transfer
      The following is a general protocol for sample preparation using 5x105 SK-N-MC cells per well in a 6-well plate:
      1. Aspirate media from cultures. Wash cells with PBS. Aspirate supernatant.
      2. Lyse cells by adding 100 μl SDS Sample Buffer per well in a 6-well plate and immediately scrape the cells off the plate and transfer the extract to a microfuge tube. Keep on ice.
      3. Sonicate for 2 s to shear DNA and reduce sample viscosity.
      4. Heat sample to 95-100°C for 5 min. Cool on ice.
      5. Microcentrifuge for 5 min.
      6. Load 20 μl onto SDS-PAGE gel (10 cm x 10 cm).
      7. Electrotransfer to nitrocellulose membrane.

      We recommend using 10 μl unphosphorylated Cdk1 protein as a negative control and 20 μl SK-N-MC cell lysate as positive control.

      Membrane Blocking, Gel and Antibody Incubations
      1. After transfer, wash membrane with 25 ml TBS for 5 min at room temperature.
      2. Incubate membrane in 25 ml of Blocking Buffer for 1-3 h at room temperature or overnight at 4°C.
      3. Wash 3 times for 5 min each with 15 ml of TBST.
      4. Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml Primary Antibody Dilution Buffer with gentle agitation overnight at 4°C.
      5. Wash 3 times for 5 min each with 15 ml of TBST.
      6. Incubate membrane with conjugated secondary antibody at the appropriate dilution in 10 ml Blocking Buffer with gentle agitation for 1 h at room temperature.
      7. Wash membrane as in step 5.

      Detection of Proteins
      Chemiluminescence.

      Variables associated with assay conditions will dictate the proper working dilution.
      Biological Information
      Immunogena synthetic phosphopeptide corresponding to amino acids surrounding the Tyr¹⁵ phosphorylation site of human Cdk1
      ImmunogenHuman
      HostRabbit
      IsotypeIgG
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Standard Handling
      Storage -20°C
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-20°C).
      Packaging Information
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      PhosphoDetect™ Anti-Cdk1 (pTyr¹⁵) Rabbit pAb MSDS

      Title

      Safety Data Sheet (SDS) 

      PhosphoDetect™ Anti-Cdk1 (pTyr¹⁵) Rabbit pAb Certificates of Analysis

      TitleLot Number
      219440

      References

      Hivatkozások áttekintése
      Norbury, C., and Nurse, P. 1992. Annu. Rev. Biochem. 61, 441.
      Hunter, T. 1995. Cell 80, 225.
      Galaktionov, K., et al. 1995. Genes Dev. 9, 1046.
      Watanabe, K., et al. 1995. EMBO J. 14, 1878.
      Atherton-Fessler, S., et al. 1993. Mol. Cell. Biol. 13, 1675.
      McGwan, C.H. and Russel, P. 1993. EMBO J. 12, 75.
      Data Sheet

      Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

      Revision26-July-2007 RFH
      ApplicationImmunoblotting (1:1000)
      Immunoprecipitation (1:100)
      Immunocytochemistry (1:100)
      Application Data
      Detection of human Cdk1 phosphorylated at Tyr15 by immunoblotting. Samples: Whole cell lysate from Saos cells left untreated (left lane) or treated with hydroxyurea (middle lane) or nocodazole (right lane). Primary antibody: PhosphoDetect™ Anti-Cdk1 (pTyr15) Rabbit pAb (Cat. No. 219440) (1:1000). Detection: chemiluminescence.
      DescriptionRabbit polyclonal antibody purified by protein A chromatography, adsorbed against non-phosphopeptide corresponding to the immunogen phosphorylation site, followed by immunoaffinity chromatography. Recognizes the ~34 kDa Cdk1 protein, phosphorylated on Tyr15.
      BackgroundEntry of all eukaryotic cells into M-phase of the cell cycle is regulated by activation of Cdk1 kinase. Activation of Cdk1 is controlled at several steps including cyclin binding and phosphorylation of threonine161. However, the critical regulatory step in activating Cdk1 during progression into mitosis appears to be dephosphorylation of Tyr15 and Thr14. Phosphorylation at Tyr15 and inhibition of Cdk1 is carried out by WEE1 and MIK protein kinases while Tyr15 dephosphorylation and activation of Cdk1 is carried out by the Cdc25 phosphatase.
      HostRabbit
      Immunogen speciesHuman
      Immunogena synthetic phosphopeptide corresponding to amino acids surrounding the Tyr¹⁵ phosphorylation site of human Cdk1
      IsotypeIgG
      Specieshuman, rat
      Positive controlSaos cells treated with hydroxyurea
      FormLiquid
      FormulationIn 150 mM NaCl, 10 mM sodium HEPES, 100 mg/ml BSA, 50% glycerol, pH 7.5.
      PreservativeNone
      CommentsAlso recognizes phosphorylated Cdk2. Does not cross-react with Cdk4, Cdk6, Cdk7, or other phosphotyrosine proteins.

      Recommended Protocol for Immunoblotting

      Solutions and Reagents
      • Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol, pH 8.5
      • SDS Sample Buffer: 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromophenol blue.
      • 10X TBS (Tris-buffered saline): To prepare 1 liter, 24.2 g Tris base, 80 g NaCl, adjust pH to 7.6 with HCl. Dilute 1:10 for use.
      • Blocking Buffer: 1X TBS, 0.1% Tween®-20 detergent with 5% non-fat dry milk.
      • Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween®-20 detergent with 5% BSA
      • Wash Buffer (TBST): 1X TBS, 0,1% Tween®-20 detergent

      Blotting Membrane
      Nitrocellulose or PVDF membranes may be used.

      SDS-PAGE and Protein Transfer
      The following is a general protocol for sample preparation using 5x105 SK-N-MC cells per well in a 6-well plate:
      1. Aspirate media from cultures. Wash cells with PBS. Aspirate supernatant.
      2. Lyse cells by adding 100 μl SDS Sample Buffer per well in a 6-well plate and immediately scrape the cells off the plate and transfer the extract to a microfuge tube. Keep on ice.
      3. Sonicate for 2 s to shear DNA and reduce sample viscosity.
      4. Heat sample to 95-100°C for 5 min. Cool on ice.
      5. Microcentrifuge for 5 min.
      6. Load 20 μl onto SDS-PAGE gel (10 cm x 10 cm).
      7. Electrotransfer to nitrocellulose membrane.

      We recommend using 10 μl unphosphorylated Cdk1 protein as a negative control and 20 μl SK-N-MC cell lysate as positive control.

      Membrane Blocking, Gel and Antibody Incubations
      1. After transfer, wash membrane with 25 ml TBS for 5 min at room temperature.
      2. Incubate membrane in 25 ml of Blocking Buffer for 1-3 h at room temperature or overnight at 4°C.
      3. Wash 3 times for 5 min each with 15 ml of TBST.
      4. Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml Primary Antibody Dilution Buffer with gentle agitation overnight at 4°C.
      5. Wash 3 times for 5 min each with 15 ml of TBST.
      6. Incubate membrane with conjugated secondary antibody at the appropriate dilution in 10 ml Blocking Buffer with gentle agitation for 1 h at room temperature.
      7. Wash membrane as in step 5.

      Detection of Proteins
      Chemiluminescence.

      Variables associated with assay conditions will dictate the proper working dilution.
      Storage -20°C
      Avoid freeze/thaw
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-20°C).
      Toxicity Standard Handling
      ReferencesNorbury, C., and Nurse, P. 1992. Annu. Rev. Biochem. 61, 441.
      Hunter, T. 1995. Cell 80, 225.
      Galaktionov, K., et al. 1995. Genes Dev. 9, 1046.
      Watanabe, K., et al. 1995. EMBO J. 14, 1878.
      Atherton-Fessler, S., et al. 1993. Mol. Cell. Biol. 13, 1675.
      McGwan, C.H. and Russel, P. 1993. EMBO J. 12, 75.