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4766
NF-κB Family Member Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

NF-κB Family Member Antibody Sampler Kit #4766

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NF-κB Family Member Antibody Sampler Kit: Image 1

Western blot analysis of extracts from control HeLa cells (lane 1) or NF-κB p65 knockout HeLa cells (lane 2) using NF-κB p65 (L8F6) Mouse mAb #6956 (upper) or β-actin (13E5) Rabbit mAb #4970 (lower). The absence of signal in the NF-κB p65 knockout HeLa cells confirms the specificity of the antibody for NF-κB p65.

NF-κB Family Member Antibody Sampler Kit: Image 2

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.

NF-κB Family Member Antibody Sampler Kit: Image 3

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

NF-κB Family Member Antibody Sampler Kit: Image 4

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

NF-κB Family Member Antibody Sampler Kit: Image 5

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.

NF-κB Family Member Antibody Sampler Kit: Image 6

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.

NF-κB Family Member Antibody Sampler Kit: Image 7

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

NF-κB Family Member Antibody Sampler Kit: Image 8

Flow cytometric analysis of HeLa cells using NF-κB2 p100/p52 (18D10) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).

NF-κB Family Member Antibody Sampler Kit: Image 9

CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across LAMC2, a known target gene of NF-κB p65 (see additional figure containing CUT&RUN-qPCR data).

NF-κB Family Member Antibody Sampler Kit: Image 10

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.

NF-κB Family Member Antibody Sampler Kit: Image 11

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).

NF-κB Family Member Antibody Sampler Kit: Image 12

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.

NF-κB Family Member Antibody Sampler Kit: Image 13

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

NF-κB Family Member Antibody Sampler Kit: Image 14

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.

NF-κB Family Member Antibody Sampler Kit: Image 15

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

NF-κB Family Member Antibody Sampler Kit: Image 16

CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 1 (upper), including LAMC2 (lower), a known target gene of NF-κB p65 (see additional figure containing CUT&RUN-qPCR data).

NF-κB Family Member Antibody Sampler Kit: Image 17

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.

NF-κB Family Member Antibody Sampler Kit: Image 18

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).

NF-κB Family Member Antibody Sampler Kit: Image 19

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.

NF-κB Family Member Antibody Sampler Kit: Image 20

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using NF-κB2 p100/p52 (18D10) Rabbit mAb.

NF-κB Family Member Antibody Sampler Kit: Image 21

CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and either NF-κB p65 (D14E12) XP® Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human LAMC2 upstream primers, and human ITM2A upstream primers. 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.

NF-κB Family Member Antibody Sampler Kit: Image 22

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

NF-κB Family Member Antibody Sampler Kit: Image 23

Immunohistochemical analysis of paraffin-embedded human osteosarcoma, using NF-κB2 p100/p52 (18D10) Rabbit mAb.

NF-κB Family Member Antibody Sampler Kit: Image 24

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.

NF-κB Family Member Antibody Sampler Kit: Image 25

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.

NF-κB Family Member Antibody Sampler Kit: Image 26

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

NF-κB Family Member Antibody Sampler Kit: Image 27

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.

NF-κB Family Member Antibody Sampler Kit: Image 28

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).

NF-κB Family Member Antibody Sampler Kit: Image 29

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

NF-κB Family Member Antibody Sampler Kit: Image 30

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)).

NF-κB Family Member Antibody Sampler Kit: Image 31

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

NF-κB Family Member Antibody Sampler Kit: Image 32

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

To Purchase # 4766T
Product # Size Price
4766T
1 Kit  (8 x 20 µl) $ 554

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
NF-κB p65 (L8F6) Mouse mAb 6956 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
B Dg Hm H Mi Mk M Pg R 65 Mouse IgG2b
RelB (C1E4) Rabbit mAb 4922 20 µl
  • WB
  • IP
H Mk M R 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
B H Mi Mk M Pg R 120 Rabbit 
NF-κB2 p100/p52 Antibody 4882 20 µl
  • WB
  • IP
H Mk M R 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
  • C&R
Dg Hm H Mk M R 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.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
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