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559304 | Anti-SAPK/JNK Rabbit pAb

559304
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      Overview

      Replacement Information

      Key Specifications Table

      Species ReactivityHostAntibody Type
      H, M, R Rb Polyclonal Antibody

      Pricing & Availability

      Catalog NumberAvailability Packaging Qty/Pack Price Quantity
      559304-200UL
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      Stocked 
      Discontinued
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      Available
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          Will advise
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          Glass bottle 200 ul
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          Description
          OverviewRecognizes the ~54 kDa SAPK/JNK protein in uv-treated HEK293 cells.
          Catalogue Number559304
          Brand Family Calbiochem®
          Application Data
          Detection of human SAPK/JNK by immunoblotting. Sample: Whole cell lysate from HEK 293 cells and SK-N-MC cells treated with UV (40 J/m2) for the indicated times. Primary antibody: Anti-SAPK/JNK Rabbit pAb (Cat. No. 559304) (1:1000). Detection: chemiluminescence.
          References
          ReferencesGupta, S., et al. 1996. EMBO J. 15(11), 2760.
          Coso, O.A., et al. 1995. Cell 81, 1137.
          Derijard, B., et al. 1994. Cell 76, 1025.
          Kyriakis, J.M., et al. 1994. Nature 369, 156.
          Hibi, M., et al. 1993. Genes Dev. 7, 2135.
          Kyriakis, J.M. and Avruch, J. 1990. J. Biol. Chem. 265, 17355.
          Product Information
          FormLiquid
          FormulationIn 150 mM NaCl, 10 mM HEPES, 50% glycerol, 0.01% BSA, pH 7.5.
          Positive controlUV treated HEK293 cells
          PreservativeNone
          Applications
          Key Applications Immunoblotting (Western Blotting)
          Immunocytochemistry
          Application NotesImmunoblotting (1:1000)
          Immunocytochemistry (1:200)
          Application CommentsRecognizes SAPK/JNK regardless of the phosphorylation state. Variables 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% 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 293 cells per well in a 6-well plate is as follows:

          1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
          2. Aspirate media from cultures; wash cells with PBS; aspirate.
          3. 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.
          4. Sonicate for 2 s to shear DNA and reduce sample viscosity.
          5. Heat sample to 95-100°C for 5 min. Cool on ice.
          6. Microcentrifuge for 5 min.
          7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
          8. Electrotransfer to nitrocellulose membrane.

          As controls, we recommend using 20 µl lysate from UV treated HEK293 cell.

          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 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 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 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 full-length, recombinant, human p54 SAPK/JNK2 fusion protein
          ImmunogenHuman
          HostRabbit
          IsotypeIgG
          Species Reactivity
          • Human
          • Mouse
          • Rat
          Antibody TypePolyclonal Antibody
          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

          SDS

          Title

          Safety Data Sheet (SDS) 

          Certificates of Analysis

          TitleLot Number
          559304

          References

          Reference overview
          Gupta, S., et al. 1996. EMBO J. 15(11), 2760.
          Coso, O.A., et al. 1995. Cell 81, 1137.
          Derijard, B., et al. 1994. Cell 76, 1025.
          Kyriakis, J.M., et al. 1994. Nature 369, 156.
          Hibi, M., et al. 1993. Genes Dev. 7, 2135.
          Kyriakis, J.M. and Avruch, J. 1990. J. Biol. Chem. 265, 17355.
          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.

          Revision15-August-2007 RFH
          ApplicationImmunoblotting (1:1000)
          Immunocytochemistry (1:200)
          Application Data
          Detection of human SAPK/JNK by immunoblotting. Sample: Whole cell lysate from HEK 293 cells and SK-N-MC cells treated with UV (40 J/m2) for the indicated times. Primary antibody: Anti-SAPK/JNK Rabbit pAb (Cat. No. 559304) (1:1000). Detection: chemiluminescence.
          DescriptionProtein A and immunoaffinity purified rabbit polyclonal antibody. Recognizes the ~54 kDa SAPK/JNK protein.
          BackgroundThe Stress-activated protein kinases (SAPK), also referred to as Jun N-terminal kinases (JNK), function in a protein kinase cascade transducing cellular stress signals. SAPK/JNKs are activated by a highly diverse group of extracellular signals, including UV light, inflammatory cytokines and a wide variety of cellular stresses. Activation occurs via phosphorylation at Thr183 and Tyr185 by the dual specificity enzyme SEK/MKK4 and as yet unidentified kinases. Since dual phosphorylation of SAPK/JNK at Thr183/Tyr185 is essential for kinase activity, phosphorylation at this site is an excellent marker of SAPK/JNK activity. Activated SAPK in turn phosphorylates and activates several transcription factors, including c-jun at Ser63/73 and ATF-2 at Ser69/71.
          HostRabbit
          Immunogen speciesHuman
          Immunogena full-length, recombinant, human p54 SAPK/JNK2 fusion protein
          IsotypeIgG
          Specieshuman, mouse, rat
          Positive controlUV treated HEK293 cells
          FormLiquid
          FormulationIn 150 mM NaCl, 10 mM HEPES, 50% glycerol, 0.01% BSA, pH 7.5.
          PreservativeNone
          CommentsRecognizes SAPK/JNK regardless of the phosphorylation state. Variables 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% 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 293 cells per well in a 6-well plate is as follows:

          1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
          2. Aspirate media from cultures; wash cells with PBS; aspirate.
          3. 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.
          4. Sonicate for 2 s to shear DNA and reduce sample viscosity.
          5. Heat sample to 95-100°C for 5 min. Cool on ice.
          6. Microcentrifuge for 5 min.
          7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
          8. Electrotransfer to nitrocellulose membrane.

          As controls, we recommend using 20 µl lysate from UV treated HEK293 cell.

          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 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 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 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 -20°C
          Avoid freeze/thaw
          Do Not Freeze Ok to freeze
          Special InstructionsFollowing initial thaw, aliquot and freeze (-20°C).
          Toxicity Standard Handling
          ReferencesGupta, S., et al. 1996. EMBO J. 15(11), 2760.
          Coso, O.A., et al. 1995. Cell 81, 1137.
          Derijard, B., et al. 1994. Cell 76, 1025.
          Kyriakis, J.M., et al. 1994. Nature 369, 156.
          Hibi, M., et al. 1993. Genes Dev. 7, 2135.
          Kyriakis, J.M. and Avruch, J. 1990. J. Biol. Chem. 265, 17355.