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400001 Anti-IκBα Rabbit pAb

400001
Purchase on Sigma-Aldrich

Panoramica

Replacement Information

Tabella delle specifiche principali

Species ReactivityHostAntibody Type
H, M, R Rb Polyclonal Antibody

Products

Numero di catalogoConfezionamento Qtà/conf
400001-100UL Fiala di plastica 100 ul
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

Documentation

Anti-IκBα Rabbit pAb MSDS

Titolo

Scheda di sicurezza (MSDS) 

Anti-IκBα Rabbit pAb Certificati d'Analisi

TitoloNumero di lotto
400001

Riferimenti bibliografici

Panoramica delle referenze
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.
Scheda tecnica

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.