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400001 Anti-IκBα Rabbit pAb

This Anti-IκBα Rabbit pAb is validated for use in Immunoblotting, Immunocytochemistry Immunoprecipitation for the detection of IκBα.

Anti-IκBα Rabbit pAb MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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400001
  
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      Overview

      Replacement Information

      Key Spec Table

      Species ReactivityHostAntibody Type
      H, M, RRbPolyclonal Antibody
      Description
      OverviewRecognizes the ~36-38 kDa phosphorylated and non-phosphorylated forms of IκBα.
      Catalogue Number400001
      Brand Family Calbiochem®
      Application Data
      Detection of human IκBα by immunoblotting. Samples: Extracts from HeLa cells treated with TNF-α. Primary antibody: Anti-IκBα Rabbit pAb (Cat. No. 400001) (1:1000). Detection: chemiluminescence.
      References
      ReferencesChen, Z. J., et al. 1996. Cell 84, 853.
      Brockman, J.A., et al. 1995. Mol. Cell. Biol. 15, 2809.
      Brown K., et al. 1995. Science 267, 1485.
      Traenckner, E.B.-M., et al. 1995. EMBO J. 14, 2876.
      Product Information
      FormLiquid
      FormulationIn 150 mM NaCl, 10 mM HEPES, 50% glycerol, 0.01% BSA, pH 7.5.
      PreservativeNone
      Quality LevelMQ100
      Applications
      Key Applications Immunoblotting (Western Blotting)
      Immunocytochemistry
      Immunoprecipitation
      Application NotesImmunoblotting (1:1000, see comments)
      Immunocytochemistry (1:2000)
      Immunoprecipitation (1:250)
      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 HeLa 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 ml SDS Sample Buffer per well 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 ml onto SDS-PAGE gel (10 cm x 10 cm).
      8. Electrotransfer to nitrocellulose membrane.

      As controls, we recommend using 20 ml of HeLa cell extracts.

      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 synthetic peptide corresponding to amino acids surrounding Ser³² of human IκBα
      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
      Global Trade Item Number
      Catalogue Number GTIN
      400001 0

      Documentation

      Anti-IκBα Rabbit pAb SDS

      Title

      Safety Data Sheet (SDS) 

      Anti-IκBα Rabbit pAb Certificates of Analysis

      TitleLot Number
      400001

      References

      Reference overview
      Chen, Z. J., et al. 1996. Cell 84, 853.
      Brockman, J.A., et al. 1995. Mol. Cell. Biol. 15, 2809.
      Brown K., et al. 1995. Science 267, 1485.
      Traenckner, E.B.-M., et al. 1995. EMBO J. 14, 2876.
      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.

      Revision02-May-2008 JSW
      ApplicationImmunoblotting (1:1000, see comments)
      Immunocytochemistry (1:2000)
      Immunoprecipitation (1:250)
      Application Data
      Detection of human IκBα by immunoblotting. Samples: Extracts from HeLa cells treated with TNF-α. Primary antibody: Anti-IκBα Rabbit pAb (Cat. No. 400001) (1:1000). Detection: chemiluminescence.
      DescriptionProtein A and immunoaffinity purified rabbit polyclonal antibody. Recognizes the ~36-38 kDa IκBα protein.
      BackgroundThe NF-κB/Rel transcription factors are present in the cytosol in an inactive state, complexed with the inhibitory IκB proteins. Activation occurs via phosphorylation of IκB-α at Ser32/36, resulting in the release and nuclear translocation of active NF-κB. IκB-α phosphorylation and resulting Rel-dependent transcription are activated by a highly diverse group of extracellular signals, including inflammatory cytokines, growth factors and chemokines. Phosphorylation of Ser32 and Ser36 has been shown to stimulate conjugation with ubiquitin followed by proteasome-mediated degradation of IκB, resulting in the release of active NF-κB.
      HostRabbit
      Immunogen speciesHuman
      Immunogena synthetic peptide corresponding to amino acids surrounding Ser³² of human IκBα
      IsotypeIgG
      Specieshuman, mouse, rat
      FormLiquid
      FormulationIn 150 mM NaCl, 10 mM HEPES, 50% glycerol, 0.01% BSA, 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 HeLa 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 ml SDS Sample Buffer per well 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 ml onto SDS-PAGE gel (10 cm x 10 cm).
      8. Electrotransfer to nitrocellulose membrane.

      As controls, we recommend using 20 ml of HeLa cell extracts.

      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 Avoid freeze/thaw
      -20°C
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-20°C).
      Toxicity Standard Handling
      ReferencesChen, Z. J., et al. 1996. Cell 84, 853.
      Brockman, J.A., et al. 1995. Mol. Cell. Biol. 15, 2809.
      Brown K., et al. 1995. Science 267, 1485.
      Traenckner, E.B.-M., et al. 1995. EMBO J. 14, 2876.