400001 | Anti-IκBα Rabbit pAb

400001
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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
      400001-100UL
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      Stocked 
      Discontinued
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      Available
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          Contact Customer Service

          Plastic ampoule 100 ul
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          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
          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

          Documentation

          SDS

          Title

          Safety Data Sheet (SDS) 

          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.

          Brochure

          Title
          Antibody Sourcebook!" Second Edition
          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 -20°C
          Avoid freeze/thaw
          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.