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442704 Anti-MAP Kinase ERK1/ERK2 Rabbit pAb

This Anti-MAP Kinase ERK1/ERK2 Rabbit pAb is validated for use in Flow Cytometry, Immunoblotting, Paraffin Sections for the detection of MAP Kinase ERK1/ERK2.

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      Übersicht

      Replacement Information

      Key Spec Table

      Host
      Rb
      Description
      OverviewRecognizes the ~42-44 kDa ERK1 and ERK2 proteins.

      This product has been discontinued.
      Catalogue Number442704
      Brand Family Calbiochem®
      Application Data
      Detection of rat MAP kinase ERK1/ERK2 by immunoblotting. Samples: Whole cell extracts from serum-induced PC12 cells. Primary antibody: Anti-MAP Kinase ERK1/ERK2 Rabbit pAb (Cat. No. 442704) (1:1000). Detection: chemiluminescence.
      References
      ReferencesHill, C.S. and Treisman, R. 1995. Cell 80, 199.
      Hunter, T. 1995. Cell 80, 225.
      Marshall, C.J. 1995. Cell 80, 179.
      Cowley, S., et al. 1994. Cell 77, 841.
      Product Information
      FormLiquid
      FormulationIn 150 mM NaCl, 10 mM HEPES, 100 µg/ml BSA, 50% glycerol, pH 7.5.
      PreservativeNone
      Applications
      Key Applications Flow Cytometry
      Immunoblotting (Western Blotting)
      Paraffin Sections
      Application NotesFlow Cytometry (1:25)
      Immunoblotting (1:1000)
      Paraffin Sections (1:100)
      Application CommentsVariables associated with assay conditions will dictate the proper working dilution.

      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.

      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 MAP kinase phosphorylation.
      2. Aspirate media. Add fresh 0.5% FBS media. Culture for 2 h.
      If cells are grown at high density, changing media before treating cells with regulator reduces basal MAP kinase phosphorylation due to factors secreted by the cells.
      3. Aspirate media. Treat cells by adding fresh 0.5% FBS media containing regulator for desired time.
      4. Aspirate media from cultures; wash cells with PBS; aspirate.
      5. Lyse cells by adding 100 µl SDS Sample Buffer per well 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 10 µl of phosphorylated (Cat. No. 454855) and nonphosphorylated
      (Cat. No. 454852) MAP kinase control proteins.

      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.
      Biological Information
      Immunogena synthetic peptide corresponding to amino acids from rat p42 MAP kinase, conjugated to KLH
      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
      Bestellnummer GTIN
      442704 0

      Documentation

      Anti-MAP Kinase ERK1/ERK2 Rabbit pAb Analysenzertifikate

      TitelChargennummer
      442704

      Literatur

      Übersicht
      Hill, C.S. and Treisman, R. 1995. Cell 80, 199.
      Hunter, T. 1995. Cell 80, 225.
      Marshall, C.J. 1995. Cell 80, 179.
      Cowley, S., et al. 1994. Cell 77, 841.
      Datenblatt

      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.

      Revision07-August-2007 RFH
      ApplicationFlow Cytometry (1:25)
      Immunoblotting (1:1000)
      Paraffin Sections (1:100)
      Application Data
      Detection of rat MAP kinase ERK1/ERK2 by immunoblotting. Samples: Whole cell extracts from serum-induced PC12 cells. Primary antibody: Anti-MAP Kinase ERK1/ERK2 Rabbit pAb (Cat. No. 442704) (1:1000). Detection: chemiluminescence.
      DescriptionRabbit polyclonal antibody purified by protein A and immunoaffinity chromatography. Recognizes the ~42-44 kDa ERK1 and ERK2 proteins.
      Backgroundp44 and p42 MAP kinases (ERK1 and ERK2) function in a protein kinase cascade that plays a critical role in the regulation of cell growth and differentiation. MAP kinases are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. Activation of MAP kinases occurs through phosphorylation of threonine and tyrosine (amino acids 202 and 204 of human MAP kinase (ERK1) at the sequence T*EY* by a single upstream MAP kinase kinase (MEK)).
      HostRabbit
      Immunogen speciesHuman
      Immunogena synthetic peptide corresponding to amino acids from rat p42 MAP kinase, conjugated to KLH
      IsotypeIgG
      Specieshamster, human, mouse, rat, zebrafish
      FormLiquid
      FormulationIn 150 mM NaCl, 10 mM HEPES, 100 µg/ml BSA, 50% glycerol, pH 7.5.
      PreservativeNone
      CommentsVariables associated with assay conditions will dictate the proper working dilution.

      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.

      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 MAP kinase phosphorylation.
      2. Aspirate media. Add fresh 0.5% FBS media. Culture for 2 h.
      If cells are grown at high density, changing media before treating cells with regulator reduces basal MAP kinase phosphorylation due to factors secreted by the cells.
      3. Aspirate media. Treat cells by adding fresh 0.5% FBS media containing regulator for desired time.
      4. Aspirate media from cultures; wash cells with PBS; aspirate.
      5. Lyse cells by adding 100 µl SDS Sample Buffer per well 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 10 µl of phosphorylated (Cat. No. 454855) and nonphosphorylated
      (Cat. No. 454852) MAP kinase control proteins.

      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.
      Storage Avoid freeze/thaw
      -20°C
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-20°C).
      Toxicity Standard Handling
      ReferencesHill, C.S. and Treisman, R. 1995. Cell 80, 199.
      Hunter, T. 1995. Cell 80, 225.
      Marshall, C.J. 1995. Cell 80, 179.
      Cowley, S., et al. 1994. Cell 77, 841.