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625725 PhosphoDetect™ Anti-TrkA (pTyr⁴⁹⁰) Rabbit pAb

This PhosphoDetect™ Anti-TrkA (pTyr⁴⁹⁰) Rabbit pAb is validated for use in Immunoblotting, Immunoprecipitation for the detection of TrkA (pTyr⁴⁹⁰).

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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      Overview

      Replacement Information

      Key Spec Table

      Host
      Rb
      Description
      Overview

      This product has been discontinued.





      Recognizes TrkA phosphorylated at Tyr490.
      Catalogue Number625725
      Brand Family Calbiochem®
      Application Data
      Detection of rat TrkA, phosphorylated on Tyr490, by immunoblotting. Samples: PC12 extracts untreated or NGF-treated. Primary antibody: PhosphoDetect™ Anti-TrkA (pTyr490) Rabbit pAb (Cat. No. 625725) (1:1000). Detection: chemiluminescence.
      References
      ReferencesSegal, R.A., and Greenberg, M.E. 1996. Annu. Rev. Neurosci. 19, 463.
      Yao, R. and Cooper, G.M. 1995. Science 267, 2003.
      Obermeier, A., et al. 1994. EMBO J. 13, 1585.
      Stephens, R.M., et al. 1994. Neuron 12, 691.
      Product Information
      FormLiquid
      FormulationIn 150 mM NaCl, 10 mM HEPES, 50% glycerol, pH 7.5.
      Applications
      Key Applications Immunoblotting (Western Blotting)
      Immunoprecipitation
      Application NotesImmunoblotting (1:1000)
      Immunoprecipitation (1:250)
      Application CommentsPhospho-specific Trk antibodies are raised against sequences within trkA, but as these sites are largely conserved between TrkB and TrkC, it is possible that this antibody will also detect activation of TrkB and TrkC. On the other hand, Trk ligands are highly specific for a given Trk receptor (e.g. NGF does not activate TrkB or TrkC).

      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% bromphenol 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.

      Protein Blotting
      A general protocol for sample preparation using 2x106 PC12 cells per well in a 6-well plate is as follows:

      1. Culture cells in medium containing 0.5% FBS for 2 days.
      We recommend plating cells directly in 0.5% FBS media to reduce basal levels of TrkA phosphorylation.
      2. Aspirate media. Add fresh media without FBS. Culture for 2 h.
      Note: if cells are grown at high density, changing media before treating cells with regulator reduces basal trkA phosphorylation due to factors secreted by cells.
      3. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
      4. Aspirate media from cultures; wash cells with PBS; aspirate.
      5. Lyse cells by adding 100 μl of SDS Sample Buffer and immediately scrape the cells off the plate and transfer the extract to a microfuge tube. Keep on ice.
      6. Sonicate for 2 s to shear DNA and reduce sample viscosity.
      7. Heat sample to 95-100°C for 5 min. Cool on ice.
      8. Microcentrifuge for 5 min.
      9. Load 20 μl onto SDS-PAGE gel (10 cm x 10 cm).
      10. Electrotransfer to nitrocellulose membrane.

      As controls, we recommend using 20 μl PC12 cell lysate.

      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 4μC.
      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.
      Biological Information
      Immunogena synthetic phosphopeptide (IENPQpYFSD) corresponding to amino acids 485-493 surrounding the Tyr⁴⁹⁰ phosphorylation site of human TrkA
      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
      Global Trade Item Number
      Catalogue Number GTIN
      625725 0

      Documentation

      PhosphoDetect™ Anti-TrkA (pTyr⁴⁹⁰) Rabbit pAb SDS

      Title

      Safety Data Sheet (SDS) 

      PhosphoDetect™ Anti-TrkA (pTyr⁴⁹⁰) Rabbit pAb Certificates of Analysis

      TitleLot Number
      625725

      References

      Reference overview
      Segal, R.A., and Greenberg, M.E. 1996. Annu. Rev. Neurosci. 19, 463.
      Yao, R. and Cooper, G.M. 1995. Science 267, 2003.
      Obermeier, A., et al. 1994. EMBO J. 13, 1585.
      Stephens, R.M., et al. 1994. Neuron 12, 691.
      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.

      Revision16-August-2007 RFH
      ApplicationImmunoblotting (1:1000)
      Immunoprecipitation (1:250)
      Application Data
      Detection of rat TrkA, phosphorylated on Tyr490, by immunoblotting. Samples: PC12 extracts untreated or NGF-treated. Primary antibody: PhosphoDetect™ Anti-TrkA (pTyr490) Rabbit pAb (Cat. No. 625725) (1:1000). Detection: chemiluminescence.
      DescriptionRabbit polyclonal antibody purified by protein A chromatography, two rounds of non-phosphopeptide affinity chromatography, and phosphopeptide immunoaffinity chromatography. Recognizes TrkA phosphorylated at Tyr490.
      BackgroundTrkA, the high affinity nerve Growth factor (NGF) receptor, autophosphor-ylates on tyrosine to activate multiple effectors. Phosphorylation at Tyr490 is required for Shc association and activation of the ras-MAP kinase cascade. Phosphorylations at Tyr674/675 (Phospho-specific antibody - Cat. No. 625730) lie within the catalytic domain and reflect trk kinase activity.
      HostRabbit
      Immunogen speciesHuman
      Immunogena synthetic phosphopeptide (IENPQpYFSD) corresponding to amino acids 485-493 surrounding the Tyr⁴⁹⁰ phosphorylation site of human TrkA
      IsotypeIgG
      Specieshuman, rat
      FormLiquid
      FormulationIn 150 mM NaCl, 10 mM HEPES, 50% glycerol, pH 7.5.
      CommentsPhospho-specific Trk antibodies are raised against sequences within trkA, but as these sites are largely conserved between TrkB and TrkC, it is possible that this antibody will also detect activation of TrkB and TrkC. On the other hand, Trk ligands are highly specific for a given Trk receptor (e.g. NGF does not activate TrkB or TrkC).

      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% bromphenol 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.

      Protein Blotting
      A general protocol for sample preparation using 2x106 PC12 cells per well in a 6-well plate is as follows:

      1. Culture cells in medium containing 0.5% FBS for 2 days.
      We recommend plating cells directly in 0.5% FBS media to reduce basal levels of TrkA phosphorylation.
      2. Aspirate media. Add fresh media without FBS. Culture for 2 h.
      Note: if cells are grown at high density, changing media before treating cells with regulator reduces basal trkA phosphorylation due to factors secreted by cells.
      3. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
      4. Aspirate media from cultures; wash cells with PBS; aspirate.
      5. Lyse cells by adding 100 μl of SDS Sample Buffer and immediately scrape the cells off the plate and transfer the extract to a microfuge tube. Keep on ice.
      6. Sonicate for 2 s to shear DNA and reduce sample viscosity.
      7. Heat sample to 95-100°C for 5 min. Cool on ice.
      8. Microcentrifuge for 5 min.
      9. Load 20 μl onto SDS-PAGE gel (10 cm x 10 cm).
      10. Electrotransfer to nitrocellulose membrane.

      As controls, we recommend using 20 μl PC12 cell lysate.

      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 4μC.
      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.
      Storage Avoid freeze/thaw
      -20°C
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-20°C).
      Toxicity Standard Handling
      ReferencesSegal, R.A., and Greenberg, M.E. 1996. Annu. Rev. Neurosci. 19, 463.
      Yao, R. and Cooper, G.M. 1995. Science 267, 2003.
      Obermeier, A., et al. 1994. EMBO J. 13, 1585.
      Stephens, R.M., et al. 1994. Neuron 12, 691.

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