![Peptide Competition and Induction Extracts prepared from ALLN pretreated Jurkat cells left unstimulated (1, 2) or stimulated with TNF-α (3-7) were resolved by SDS-PAGE on a 10% polyacrylamide
gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer for one hour at room temperature and incubated with anti-IκBα pan (1, 3) or IκBα [pSpS32/36] antibody (2, 4-7) overnight at 4∞C in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1-4), the non-phosphopeptide corresponding to the immunogen (5), a generic phosphoserine-containing peptide (6), or, the
phosphopeptide immunogen (7). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate and bands were
detected using the Pierce SuperSignal‘ method.
The data show that the phosphorylation of IκBα is induced by the addition of TNF-α and that the peptide corresponding to IκBα [pSpS32/36] blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that treatment with TNF-α leads to degradation of IκBα protein (compare lanes 1 and 3)](/media/images/0092804.jpeg)
BP7089 I Kappa B alpha (IKBa) pSer32/36 antibody
see related secondary antibodies
0.1 ml / US$ 495
NOVUS BIOLOGICALS
PO Box 802 Littleton, CO 80160, USA
E-Mail: novus@novus-biologicals.com
Homepage: http://www.novus-biologicals.com
PO Box 802 Littleton, CO 80160, USA
E-Mail: novus@novus-biologicals.com
Homepage: http://www.novus-biologicals.com
Quick Overview
Rabbit anti I Kappa B alpha (IKBa) pSer32/36 -
Synonyms
IkappaB alpha,
Product Description
Rabbit anti I Kappa B alpha (IKBa) pSer32/36 -, Presentation: Aff - Purified. Product is tested for Western blot / Immunoblot ( WB )
Properties
| Applications | Western blot / Immunoblot ( WB ) |
| Reactivity | Human ( Hu ), Mouse ( Ms ), Rat ( Rt ), Por ( Por ), Chicken ( Chk ) |
| Presentation | Aff - Purified |
| Host | Rabbit |
| Catalog Number | BP7089 |
| Quantity | 0.1 ml |
| Price | US$ 495 |
| Delivery | Worldwide |
| Manufacturer | Acris Antibodies GmbH |
| Datasheet | view  BP7089.pdf |
Datasheet Extract
| Polyclonal Antibody to IκBα [pSpS32/36] //Phosphospecific Antibody | |
| Background | Inhibitor of NFκB α (IκBα) is a ~40 kDa protein that has a central role in the regulation of transcription mediated by the NFκB transcription factor. In the absence of a stimulus, NFκB is sequestered in the cytoplasm through binding to IκB proteins. A variety of stimuli (e.g., proinflammatory cytokines, Tumor Necrosis Factor-α, and interleukin-1β) lead to phosphorylation and activation of the IKK complex (consisting of IKKα, β, and γ). The activated IKK complex in turn phosphorylates IκBα serines 32 and 36, leading to its subsequent ubiquitination and degradation by the proteasome. The free NFκB translocates to the nucleus and stimulates transcription of NFκB-responsive genes. Phosphorylation of these serine residues by several other kinases, including p90S6 kinase (Rsk1), NFκB-Inducing Kinase (NIK), and Mitogen-Activated Protein Kinase Kinase Kinase-1 (MEKK1), with subsequent activation of NFκB-mediated transcription, has also been reported. A component of the NFκB complex, RelA, has recently been shown to regulate the level of IκBα phosphorylation. |
| Immunogen | The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human IκBα that contains serines 32 and 36.
|
| Format | State: Liquid Ig fraction Purification: Peptide affinity chromatography. Purified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively pre-adsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated I?B?. The final product is generated by affinity chromatography using a I?B?-derived peptide that is phosphorylated at serines 32 and 36. BufferSystem: Dulbecco's phosphate buffered saline (without Mg2+ and Ca2+), pH 7.3 (+/- 0.1), 50% glycerol, with 1.0 mg/mL BSA (IgG, protease free) as a carrier and 0.05% sodium azide as preservative. |
| Applications | The antibody has been used for Western blotting applications. For Western blotting applications, we recommend using the antibody at a 1:1000 dilution. Positive control used: Jurkat cells pretreated for 30 minutes with N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) and stimulated with TNF-α. |
| Specificity | This antibody reacts with IκBα. Species: Human. |
| Add. Information | Mouse and pig (100% homologous), rat and chicken (93% homologous) IκBα have not been tested, but are expected to react. |
| Storage | Store at 4°C short term only. Aliquot and store at -20°C to -80°C for longer. Avoid repeated freezing and thawing. Shelf life: one year from despatch. |
| References | Ding, G.R., et al. (2003) Radiosensitization by inhibition of IκBα phosphorylation in human glioma cells. Radiat. Res. 160(2):232-237.
Miskolci, V., et al. (2003) Okadaic acid induces sustained activation of NFκB and degradation of the nuclear IκBα in human neutrophils. Arch. Biochem. Biophys. 417(1):44-52. Yang, L., et al. (2003) RelA control of IκBα phosphorylation: a positive feedback loop for high affinity NF-κB complexes. J. Biol. Chem. 278(33):30881-30888. Ghosh, S. and M. Karin. (2002) Missing pieces of the NF-κB puzzle. Cell 109(Suppl.):S81-S96. Carcamo, J.M., et al. (2002) Vitamin A suppresses TNFα-induced NF-κB activation by inhibiting IκBα phosphorylation. Biochem. 41(43):12995-13002. Pandey, S.K., et al. (2002) Wortmannin-sensitive pathway is required for insulin-stimulated phosphorylation of Inhibitor κBα. Endocrin. 143(2):375-385. Chen, F., et al. (2001) New insights into the role of Nuclear Factor-κB in cell growth regulation. Am. J. Pathol. 159(2):387-397. Li, X., et al. (1999) Characterization of NFκB activation by detection of green fluorescent proteintagged IκB degradation in living cells. J. Biol. Chem. 274(30):21244-21250. DiDonato, J., et al. (1997) Mapping of the inducible IκB phosphorylation sites that signal its ubiquitination and degradation. Mol. Cell. Biol. 16(4):1295-1304. |
| Protocols | Western Blotting Procedure
1. Lyse approximately 10e7 cells in 0.5 mL of ice cold Cell Lysis Buffer (formulation provided below). This buffer, a modified RIPA buffer, is suitable for recovery of most proteins, including membrane receptors, cytoskeletal-associated proteins, and soluble proteins. This cell lysis buffer formulation is available as a separate product which requires supplementation with protease inhibitors immediately prior to use. Other cell lysis buffer formulations, such as Laemmli sample buffer and Triton-X 100 buffer, are also compatible with this procedure. Additional optimization of the cell stimulation protocol and cell lysis procedure may be required for each specific application. 2. Remove the cellular debris by centrifuging the lysates at 14,000 x g for 10 minutes. Alternatively, lysates may be ultracentrifuged at 100,000 x g for 30 minutes for greater clarification. 3. Carefully decant the clarified cell lysates into clean tubes and determine the protein concentration using a suitable method, such as the Bradford assay. Polypropylene tubes are recommended for storing cell lysates. 4. React an aliquot of the lysate with an equal volume of 2x Laemmli Sample Buffer (125 mM Tris, pH 6.8, 10% glycerol, 10% SDS, 0.006% bromophenol blue, and 130 mM dithiothreitol [DTT]) and boil the mixture for 90 seconds at 100oC. 5. Load 10-30 μg of the cell lysate into the wells of an appropriate single percentage or gradient minigel and resolve the proteins by SDS-PAGE. 6. In preparation for the Western transfer, cut a piece of PVDF membrane slightly larger than the gel. Soak the membrane in methanol for 1 minute, then rinse with ddH2O for 5 minutes. 7. Soak the membrane, 2 pieces of Whatman paper, and Western apparatus sponges in transfer buffer (formulation provided below) for 2 minutes. 8. Assemble the gel and membrane into the sandwich apparatus. 9. Transfer the proteins at 140 mA for 60-90 minutes at room temperature. 10. Following the transfer, rinse the membrane with Tris buffered saline for 2 minutes. 11. Block the membrane with blocking buffer (formulation provided below) overnight at 4°C. 12. Incubate the blocked blot with primary antibody at a 1:1000 starting dilution in Tris buffered saline supplemented with 3% BSA and 0.1% Tween 20 for 2 hours at room temperature. 13. Wash the blot with several changes of Tris buffered saline supplemented with 0.1% Tween 20. 14. Detect the antibody band using an appropriate secondary antibody, such as goat F(ab)2 anti-rabbit IgG alkaline phosphatase conjugate or goat F(ab)2 anti-rabbit IgG horseradish peroxidase conjugate in conjunction with your chemiluminescence reagents and instrumentation. BUFFER FORMULATIONS Cell Lysis Buffer: 10 mM Tris, pH 7.4 100 mM NaCl 1 mM EDTA 1 mM EGTA 1 mM NaF 20 mM Na4P2O7 2 mM Na3VO4 0.1% SDS 0.5% sodium deoxycholate 1% Triton-X 100 10% glycerol 1 mM PMSF (made from a 0.3 M stock in DMSO) or 1 mM AEBSF (water soluble version of PMSF) 60 µg/mL aprotinin 10 µg/mL leupeptin 1 mg/mL pepstatin (alternatively, protease inhibitor cocktail such as Sigma catalog number P2714 may be used) Transfer Buffer 2.4 gm Tris base 14.2 gm glycine 200 mL methanol Q.S. to 1 liter, then add 1 mL 10% SDS. Cool to 4oC prior to use. Tris Buffered Saline 20 mM Tris-HCl, pH 7.4 0.9% NaCl Blocking Buffer Formulation: 100 mL Tris buffered saline 5 gm BSA 0.1 mL Tween 20 Peptide Competition Experiment Phosphorylation Site Specific Antibodies (PSSAs) have been developed to enable the specific and sensitive detection of phosphorylation of particular amino acid residues in target proteins, while circumventing the need for protein purification, phosphopeptide mapping or handling radioactivity. The specificity of a PSSA in each experimental system can be confirmed through peptide competition. In this technique, aliquots of antibody are pre-incubated with peptide containing the sequence of the phosphopeptide immunogen used to raise the PSSA and the corresponding non-phosphopeptide. Following preincubation with the peptide, each antibody preparation is then used as a probe in antibody-based detection methods, such as Western blotting, immunocytochemistry, flow cytometry, or ELISA. With a PSSA specific for the phosphorylated target protein, pre-incubation with an excess of peptide containing the sequence of the phosphopeptide immunogen will block all antigen binding sites, while pre-incubation with the corresponding non-phosphopeptide will not affect the antibody. In performing the Peptide Competition Experiment, it is important to note that the optimal dilutions of both antibody and peptide should be determined empirically for each specific application. The optimal dilution of antibody in these procedures is below saturating, as determined by previous experiments in your system. If an optimal antibody dilution has not been determined in your system, please refer to the Suggested Working Dilution on the antibody Product Analysis Sheet for guidance on an appropriate starting dilution. The optimal dilution of peptide used in these procedures will depend on the overall affinity or avidity of the antibody, as well as the quantity of the target antigen. A 50-150 fold molar excess of peptide to antibody is found to be effective for most peptide competition experiments. In the example presented below, the PSSA is used as a dilution of 1:1000 and the peptides are used at a concentration of 333 nM. The total volume of the phosphopeptide and non-phosphopeptide-pre-incubated antibody preparations is 2 mL, sufficient for probing Western blot strips, as well as for use in other antibody-based detection methods. Under these conditions, the molar excess of peptide to antibody is > / = 50. Procedure: 1. Prepare three identical test samples, such as identical PVDF or nitrocellulose strips to which the protein of interest has been transferred. The test samples should be blocked using a blocking buffer, such as Tris buffered saline supplemented with 0.1% Tween 20, and either 5% BSA or 5% non-fat dried milk. 2. Prepare 6.5 mL of working antibody stock solution (1:1000 in this example) by adding 6.5 μL of antibody stock solution to 6.5 mL of buffer containing blocking protein, such as TBS supplemented with 0.1% Tween 20, and either 3% BSA or 3% non-fat dried milk. 3. Apportion the unused PSSA into working aliquots and store at ï?20°C for future use (the stock PSSA contains 50% glycerol and will not freeze at this temperature). 4. Allow the lyophilized control peptides to reach room temperature, ideally under desiccation. 5. Reconstitute each of the control peptides (supplied at 0.1 mg/vial) to a concentration of 66.7 μM with nanopure water. For a peptide with a molecular mass of 1500 (stated on the peptide Product Analysis Sheet), reconstitution with 1 mL water yields a solution with a concentration of 66.7 μM. 6. Apportion the unused reconstituted peptide solutions into working aliquots and store at -20°C for future use. 7. Label 3 test tubes as follows: tube 1: water only no peptide control tube 2: phosphopeptide tube 3: non-phosphopeptide 8. Into each tube, pipette the following components tube 1: 2 mL diluted PSSA solution plus 10 μL nanopure water tube 2: 2 mL diluted PSSA solution plus 10 μL phosphopeptide tube 3: 2 mL diluted PSSA solution plus 10 μL non-phosphopeptide 9. Incubate the three tubes for 30 minutes at room temperature with gentle rocking. During this incubation, the peptides have the chance to bind to the combining site of the antibody. 10. At the end of the incubation step, transfer the contents of each of the three tubes to clean reaction vessels containing one of the three identical test samples. For Western blotting strips: Incubate the strips with the pre-incubated antibody preparations for 1 hour at room temperature or overnight at 4°C. Wash each strip four times, five minutes each, to remove unbound antibody. Transfer each strip to a new solution containing a labeled secondary antibody [e.g., goat F(ab)2 anti-rabbit IgG alkaline phosphatase conjugate or goat F(ab)2 anti-rabbit IgG horseradish peroxidase conjugate. Remove unbound secondary antibody by thorough washing, and develop the signal using your chemiluminescent reagents and instrumentation. The signal obtained with antibody incubated with the Water Only, No Peptide Control (Tube 1), represents the maximum signal in the assay. This signal should be eliminated by pre-incubation with the Phosphopeptide (Tube 2), while pre-incubation with the Non-Phosphopeptide (Tube 3) should not impact the signal. If the Phosphopeptide only partially eliminates the signal, repeat the procedure using twice the volume of water or peptide solutions listed in Step 8. If partial competition is seen following pre-incubation with the Non-Phosphopeptide, repeat the procedure using half the volumes of water or peptide solutions listed in Step 8. |
| Pictures | Peptide Competition and Induction Extracts prepared from ALLN pretreated Jurkat cells left unstimulated (1, 2) or stimulated with TNF-α (3-7) were resolved by SDS-PAGE on a 10% polyacrylamide
gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer for one hour at room temperature and incubated with anti-IκBα pan (1, 3) or IκBα [pSpS32/36] antibody (2, 4-7) overnight at 4â??C in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1-4), the non-phosphopeptide corresponding to the immunogen (5), a generic phosphoserine-containing peptide (6), or, the phosphopeptide immunogen (7). After washing, membranes were incubated with goat F(abâ??)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignalâ?? method. The data show that the phosphorylation of IκBα is induced by the addition of TNF-α and that the peptide corresponding to IκBα [pSpS32/36] blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that treatment with TNF-α leads to degradation of IκBα protein (compare lanes 1 and 3) |
12 Secondary Antibodies
| Catalog No. | Name / Host | Presentation | Reactivity | ||||
|---|---|---|---|---|---|---|---|
| R1364B | Rabbit IgG (H&L) | ||||||
| Goat | Biotin | 2 mg / US$ 295 | |||||
| R1364F | Rabbit IgG (H&L) | ||||||
| Goat | FITC | 2 mg / US$ 285 | |||||
| R1364T | Rabbit IgG (H&L) | ||||||
| Goat | TRITC | 2 mg / US$ 285 | |||||
| R1364TR | Rabbit IgG (H&L) | ||||||
| Goat | Texas Red | 2 mg / US$ 295 | |||||
| R1364HRP | Rabbit IgG (H&L) | ||||||
| Goat | HRP | 2 mg / US$ 295 | |||||
| R1364AP | Rabbit IgG (H&L) | ||||||
| Goat | AP | 1 mg / US$ 325 | |||||
| R1458C2 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy2 | 1 mg / US$ 445 | |||||
| R1458C3 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3 | 1 mg / US$ 445 | |||||
| R1458C35 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3.5 | 1 mg / US$ 445 | |||||
| R1458C5 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5 | 1 mg / US$ 445 | |||||
| R1458C55 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5.5 | 1 mg / US$ 445 | |||||
| R1427R | Rabbit IgG (H&L) F(ab')2 Fragment multi-species ads. | ||||||
| Donkey | PE | 1 ml / US$ 455 | |||||
Click here to see all secondary antibodies for 'BP7089 I Kappa B alpha (IKBa) pSer32/36'.