Revision 1
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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UniProt ID:

#P25963

Entrez-Gene Id:

4792

Product Information

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 detects endogenous levels of total IκBα protein. Phospho-IκBα (Ser32/36) (5A5) Mouse mAb #9246 detects endogenous levels of IκBα only when phosphorylated at Ser32/36. Neither antibody cross-reacts with other IκB family members at physiological levels.

Source / Purification

IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 is produced by immunizing mice with a GST-IκBα fusion protein corresponding the amino-terminus of human IκBα. Phospho-IκBα (Ser32/36) (5A5) Mouse mAb #9246 is produced by immunizing mice with a synthetic phosphopeptide corresponding to residues surrounding Ser32/36 of human IκBα. Total cell extracts from HeLa cells prepared without treatment serve as a negative control. Supplied in SDS Sample Buffer. Total cell extracts from HeLa cells prepared with TNF-α treatment (#2169, 20 ng/ml for 5 minutes) serve as a positive control. Supplied in SDS Sample Buffer.

Product Description

The PhosphoPlus® IκBα (Ser32/36) Antibody Kit provides reagents and protocols for the rapid analysis of IκBα phosphorylation at Ser32/36. The kit contains a total IκBα antibody, a phospho-specific antibody, and positive/negative control whole cell lysates, along with secondary antibodies and reagents for Western blotting.

Background

The NF-κB/Rel transcription factors are present in the cytosol in an inactive state complexed with the inhibitory IκB proteins (1-3). Activation occurs via phosphorylation of IκBα at Ser32 and Ser36 followed by proteasome-mediated degradation that results in the release and nuclear translocation of active NF-κB (3-7). IκBα phosphorylation and resulting Rel-dependent transcription are activated by a highly diverse group of extracellular signals including inflammatory cytokines, growth factors, and chemokines. Kinases that phosphorylate IκB at these activating sites have been identified (8).

  1. Baeuerle, P.A. and Baltimore, D. (1988) Science 242, 540-6.
  2. Beg, A.A. and Baldwin, A.S. (1993) Genes Dev 7, 2064-70.
  3. Finco, T.S. et al. (1994) Proc Natl Acad Sci USA 91, 11884-8.
  4. Brown, K. et al. (1995) Science 267, 1485-8.
  5. Brockman, J.A. et al. (1995) Mol Cell Biol 15, 2809-18.
  6. Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.
  7. Chen, Z.J. et al. (1996) Cell 84, 853-62.
  8. Karin, M. and Ben-Neriah, Y. (2000) Annu Rev Immunol 18, 621-63.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PhosphoPlus is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 1
#9240

PhosphoPlus® IκBα (Ser32/36) Antibody Kit

PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 1 Expand Image
Western blot analysis of extracts from various cell lines using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 2 Expand Image
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 3 Expand Image
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO* is added and emits light during enzyme catalyzed decomposition.
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 4 Expand Image
Western blot analysis of NF-κB Control Cell Extracts #9243, using IKKα Antibody #2682 (upper), IKKβ (2C8) Rabbit mAb #2370 (middle) and Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb #2697 (lower).
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 5 Expand Image
Western blot analysis of extracts from NIH/3T3 cells, untreated or TNF-α-treated (#8902, 20 ng/ml) for 5 minutes, using Phospho-IκBα (Ser32/36) (5A5) Mouse mAb #9246 (upper) or IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814, and β-Actin (D6A8) Rabbit mAb #8457 (lower).
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 6 Expand Image
Western blot analysis of extracts from THP-1 cells, differentiated with TPA (#9905, 80 nM for 24h) and treated with 1 μg/ml LPS for the indicated times, using Phospho-IκBα (Ser32/36) (5A5) Mouse mAb #9246 (upper) and IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) (lower).
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 7 Expand Image
Western blot analysis of NF-κB Control Cell Extracts #9243, using IκBα (44D4) Rabbit mAb #4812 (left) and Phospho-IκBα (Ser32) (14D4) Rabbit mAb #2859 (right).
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 8 Expand Image
Western blot analysis of extracts from various cell lines, untreated or treated with IFNa (#36000, 20 ng/mL, 5 min); using Phospho-IκBα (Ser32/36) (5A5) Mouse mAb #9246 (upper), IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 9 Expand Image
Immunoprecipitation of IkBa from HeLa cell extracts. Lane 1 is 10% input, lane 2 is precipitated with Mouse (G3A1) mAb IgG1 Isotype Control #5415, and lane 3 is IκBα (L35A5) Mouse mAb (Amino-terminal Antigen), #4814. Western blot was performed using IκBα Antibody #9242.
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 10 Expand Image
Western blot analysis of extracts from THP-1 cells, differentiated with TPA (#9905, 80 nM for 24 h) and treated with 1 μg/ml LPS for the indicated times, using Phospho-IκBα (Ser32/36) (5A5) Mouse mAb (upper) and IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 (lower).
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 11 Expand Image
Immunohistochemical analysis of paraffin-embedded human leiomyoma, using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen).
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 12 Expand Image
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen).
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 13 Expand Image
Immunohistochemical analysis of paraffin-embedded human renal adenocarcinoma, using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen).
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 14 Expand Image
Confocal immunofluorescent analysis of HeLa cells, untreated (left), or TNF-α-treated (#8902, 10 ng/ml for 15 min, right) using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
PhosphoPlus® IκBα (Ser32/36) Antibody Kit: Image 15 Expand Image
Flow cytometric analysis of NIH/3T3 cells, treated with TNF-α (#8902, 10 ng/ml for 5 min; blue, negative) or untreated (green, positive) using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) (solid lines) or concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.