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4766
NF-κB Family Member Antibody Sampler Kit
Primary Antibodies

NF-κB Family Member Antibody Sampler Kit #4766

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Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (30 ng/ml, 1 hr) and either NF-κB p65 (L8F6) Mouse mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human IκBα Promoter Primers #5552, human IL-8 promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Western blot analysis of extracts from Raji, THP-1 and BaF3 cells using RelB (C1E4) Rabbit mAb.

Western blot analysis of extracts from various cell lines and tissues using c-Rel (D4Y6M) Rabbit mAb.

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (30 ng/ml) for 1 hour and either NF-κB1 p105/p50 (D7H5M) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by Real-Time PCR using SimpleChIP® Human IκBα Promoter Primers #5552, human IAP2 promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

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 NF-κB1 p105 Antibody.

Western blot analysis of extracts from THP-1, L929, C6, and COS cells, using NF-kappaB2 p100 Antibody.

Flow cytometric analysis of HeLa cells using NF-kappaB2 p100/p52 (18D10) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across IL-8, a known target gene of NFκB (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

Flow cytometric analysis of HeLa cells using NF-κB p65 (D14E12) XP® Rabbit mAb (blue) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red).

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.

Flow cytometric analysis of HeLa cells using NF-κB p65 (L8F6) Mouse mAb (blue) compared to a nonspecific negative control antibody (red).

Western blot analysis of extracts from Neuro-2a cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® c-Rel siRNA I (Mouse Specific) #13058 (+) or SignalSilence® c-Rel siRNA II (Mouse Specific) #13170 (+), using c-Rel (D4Y6M) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The c-Rel (D4Y6M) Rabbit mAb confirms silencing of c-Rel expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.

Western blot analysis of extracts from various cell lines using NF-κB1 p105/p50 (D7H5M) Rabbit mAb.

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using NF-kappaB2 p100/p52 (18D10) Rabbit mAb.

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and either NF-κB p65 (D14E12) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by Real-Time PCR using SimpleChIP® Human IκBα Promoter Primers #5552, human IL-8 promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/mL, 20 min; right), using NF-κB p65 (L8F6) Mouse mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Western blot analysis of extracts from HeLa cells, untreated (-) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (10 ng/ml) for the indicated times, using NF-κB1 p105/p50 (D7H5M) Rabbit mAb.

Immunohistochemical analysis of paraffin-embedded human osteosarcoma, using NF-kappaB2 p100/p52 (18D10) Rabbit mAb.

Immunohistochemical analysis of human chronic cholecystitis tissue using NF-κB p65 (L8F6) Mouse mAb.

Western blot analysis of extracts from HeLa, and COS cells, using NF-kB2 p100/p52 (18D10) Rabbit mAb.

Confocal immunofluorescent analysis of HT-1080 cells, untreated (left) or treated with hTNF-α #8902 (20 ng/ml, 20 min) (right), using NF-κB p65 (D14E12) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Immunohistochemical analysis of paraffin-embedded OVCAR8 cell pellets treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (left) or treated with SignalSilence® NF-κB p65 siRNA I #6261 (right), using NF-κB p65 (L8F6) Mouse mAb.

Immunohistochemical analysis using NF-κB p65 (D14E12) XP® Rabbit mAb on SignalSlide® NF-κB p65 IHC Controls #12873 (paraffin-embedded HCT116 cells, untreated (left) or treated with hTNF-α #8902 (right)).

Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, untreated (left) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (right), using NF-κB p65 (L8F6) Mouse mAb.

Immunohistochemical analysis of paraffin-embedded human chronic cholecystitis using NF-κB p65 (D14E12) XP® Rabbit mAb.

Western blot analysis of HeLa cell extracts, untreated (-) or NF-κB p65 knock-out (+), using NF-κB p65 (L8F6) Mouse mAb #6956 (upper) or β-actin (13E5) Rabbit mAb #4970 (lower).

Western blot analysis of HeLa cell extracts, untreated (-) or NF-κB p65 knock-out (+), using NF-κB p65 (D14E12) XP® Rabbit mAb, #8242 (upper) or β-actin (13E5) Rabbit mAb #4970 (lower).

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® NF-κB p65 siRNA I #6261 (+), using NF-κB p65 (L8F6) Mouse mAb (upper) or α-Tubulin (11Η10) Rabbit mAb #2125 (lower). The NF-κB p65 (L8F6) Mouse mAb confirms silencing of NF-κB p65 expression, while the α-Tubulin (11Η10) Rabbit mAb is used as a loading control.

Western blot analysis of extracts from various cell lines using NF-κB p65 (D14E12) XP® Rabbit mAb.

Western blot analysis of extracts from various cell lines using NF-κB p65 (L8F6) Mouse mAb.

To Purchase # 4766T
Product # Size Price
4766T
1 Kit  (8 x 20 µl) $ 538

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
NF-κB p65 (L8F6) Mouse mAb 6956 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H M R Hm Mk Mi B Dg Pg 65 Mouse IgG2b
RelB (C1E4) Rabbit mAb 4922 20 µl
  • WB
  • IP
H M R Mk 70 Rabbit IgG
c-Rel (D4Y6M) Rabbit mAb 12707 20 µl
  • WB
H M R 68-78 Rabbit IgG
NF-κB1 p105/p50 (D7H5M) Rabbit mAb 12540 20 µl
  • WB
  • IP
  • ChIP
H M 50 Active form. 120 Precursor Rabbit IgG
NF-κB1 p105 Antibody 4717 20 µl
  • WB
  • IP
H M R Mk Mi B Pg 120 Rabbit 
NF-κB2 p100/p52 Antibody 4882 20 µl
  • WB
  • IP
H M R Mk 52 (mature). 120 (precursor). Rabbit 
NF-κB2 p100/p52 (18D10) Rabbit mAb 3017 20 µl
  • WB
  • IHC
  • F
H Mk 52 active form. 120 precursor. Rabbit IgG
NF-κB p65 (D14E12) XP® Rabbit mAb 8242 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H M R Hm Mk Dg 65 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

Product Description

This kit contains reagents to examine total protein levels of the five NF-κB/Rel family members: p65/RelA, RelB, c-Rel, NF-κB1 (p105/p50) and NF-κB2 (p100/p52).

Specificity / Sensitivity

Each antibody in this kit recognizes endogenous levels of its target protein regardless of post-translational modification state such as phosphorylation or acetylation. The NF-κB1 p105/p50 (D7H5M) Rabbit mAb #12540 detects both the precursor protein p105 and its cleavage product p50, while the NF-κB1 p105 Antibody #4717 only detects p105 and will not cross-react with p50. Both the NF-κB2 p100/p52 Antibody #4882 and the NF-κB2 p100/p52 (18D10) Rabbit mAb (Human Specific) #3017 will cross-react with the precursor protein p100 and its cleavage product p52.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to amino acid residues near the carboxy terminus of human NF-κB p65, surrounding Ser424 of human RelB, surrounding Leu65 of human c-Rel protein, surrounding Gly415 of human NF-κB p105/p50 protein, surrounding Glu498 of human NF-κB p65/RelA protein, and near the amino terminus of human NF-κB2 p100/p52. Polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to amino acid residues at the the carboxy terminus of human NF-κB1 p105 and near the amino terminus of human NF-κB2 p100/p52. Polyclonal antibodies are purified by Protein A and peptide affinity chromatography.

Background

Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, c-Rel, RelB, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Both p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. Rel proteins bind p50 and p52 to form dimeric complexes that bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins (3-5). NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKKα (IKK1) regulate the phosphorylation and processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus (9-11).

  1. Baeuerle, P.A. and Henkel, T. (1994) Annu Rev Immunol 12, 141-79.
  2. Baeuerle, P.A. and Baltimore, D. (1996) Cell 87, 13-20.
  3. Haskill, S. et al. (1991) Cell 65, 1281-9.
  4. Thompson, J.E. et al. (1995) Cell 80, 573-82.
  5. Whiteside, S.T. et al. (1997) EMBO J 16, 1413-26.
  6. Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.
  7. Scherer, D.C. et al. (1995) Proc Natl Acad Sci USA 92, 11259-63.
  8. Chen, Z.J. et al. (1996) Cell 84, 853-62.
  9. Senftleben, U. et al. (2001) Science 293, 1495-9.
  10. Coope, H.J. et al. (2002) EMBO J 21, 5375-85.
  11. Xiao, G. et al. (2001) Mol Cell 7, 401-9.
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.