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55764
NF-κB Family Antibody Sampler Kit II
Primary Antibodies
Antibody Sampler Kit

NF-κB Family Antibody Sampler Kit II #55764

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NF-κB Family Antibody Sampler Kit II: Image 1

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 Antibody Sampler Kit II: Image 2

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 Antibody Sampler Kit II: Image 3

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 Antibody Sampler Kit II: Image 4

Flow cytometric analysis of HeLa cells, untreated (blue) or TNF-α-treated (green), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb compared to a nonspecific negative control antibody (red).

NF-κB Family Antibody Sampler Kit II: Image 5

Chromatin immunoprecipitations were performed with cross-linked chromatin from HDLM-2 cells and either RelB (D7D7W) 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 human IAP2 promoter primers, SimpleChIP® Human IκBα Promoter Primers #5552, 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 Antibody Sampler Kit II: Image 6

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

NF-κB Family Antibody Sampler Kit II: Image 7

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-κB1 p105/p50 (D4P4D) 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 Antibody Sampler Kit II: Image 8

Chromatin immunoprecipitations were performed with cross-linked chromatin from HDLM-2 cells and either NF-κB2 p100/p52 (D7A9K) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. 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 Antibody Sampler Kit II: Image 9

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 Antibody Sampler Kit II: Image 10

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 Antibody Sampler Kit II: Image 11

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 Antibody Sampler Kit II: Image 12

Confocal immunofluorescent analysis of HeLa cells, serum starved (left) or TNF-α treated (#8902 at 20 ng/ml for 20 min, right), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® phalloidin 555 (red).

NF-κB Family Antibody Sampler Kit II: Image 13

Flow cytometric analysis of LNCaP cells (blue) and HDLM-2 cells (green) using RelB (D7D7W) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

NF-κB Family Antibody Sampler Kit II: 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 Antibody Sampler Kit II: Image 15

Flow cytometric analysis of C2C12 cells using NF-κB1 p105/p50 (D4P4D) Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.

NF-κB Family Antibody Sampler Kit II: Image 16

Immunoprecipitation of NF-κB2 p100/p52 from HDLM-2 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is NF-κB2 p100/p52 (D7A9K) Rabbit mAb. Western blot analysis was performed using NF-κB2 p100/p52 (D7A9K) Rabbit mAb. A conformation-specific secondary antibody was used to avoid cross-reactivity with IgG.

NF-κB Family Antibody Sampler Kit II: Image 17

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 Antibody Sampler Kit II: Image 18

Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated or TNF-α treated (#2169, 20 ng/ml for 5 minutes), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (upper) or NF-κB p65 Antibody #3034 (lower).

NF-κB Family Antibody Sampler Kit II: Image 19

Confocal immunofluorescent analysis of PANC-1 cells (left, positive) and Kelly cells (right, negative) using RelB (D7D7W) Rabbit mAb (green). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

NF-κB Family Antibody Sampler Kit II: Image 20

Confocal immunofluorescent analysis of C2C12 cells, untreated (left) or treated with Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, 30 min; right), using NF-κB1 p105/p50 (D4P4D) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).

NF-κB Family Antibody Sampler Kit II: Image 21

Western blot anlaysis of extracts from various cell lines using NF-κB2 p100/p52 (D7A9K) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.

NF-κB Family Antibody Sampler Kit II: Image 22

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-NF-κB p65 (Ser536) (93H1) Rabbit mAb (upper) and NF-κB p65 (C22B4) Rabbit mAb #4764 (lower).

NF-κB Family Antibody Sampler Kit II: Image 23

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using RelB (D7D7W) Rabbit mAb.

NF-κB Family Antibody Sampler Kit II: Image 24

Immunoprecipitation of NF-κB1 p105/p50 from Raji cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or NF-κB1 (D4P4D) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using NF-κB1 p105/p50 (D4P4D) Rabbit mAb. Mouse Anti-rabbit IgG (Light-Chain Specific) (L57A3) mAb #3677 was used as a secondary antibody.

NF-κB Family Antibody Sampler Kit II: Image 25

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using RelB (D7D7W) Rabbit mAb.

NF-κB Family Antibody Sampler Kit II: Image 26

Western blot analysis of extracts from various cell lines and rat spleen using NF-κB1 p105/p50 (D4P4D) Rabbit mAb.

NF-κB Family Antibody Sampler Kit II: Image 27

Immunohistochemical analysis of paraffin-embedded HDLM-2 (left) and LNCaP (right) cell pellets using RelB (D7D7W) Rabbit mAb.

NF-κB Family Antibody Sampler Kit II: 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 Antibody Sampler Kit II: Image 29

Immunohistochemical analysis of paraffin-embedded human tonsil using RelB (D7D7W) Rabbit mAb.

NF-κB Family Antibody Sampler Kit II: 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 Antibody Sampler Kit II: Image 31

Immunoprecipitation of RelB from Raji cell extracts. Lane 1 represents 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 and lane 3 is RelB (D7D7W) Rabbit mAb. Western blot was performed using RelB (D7D7W) Rabbit mAb. A conformation specific secondary antibody was used to avoid cross reativity with IgG.

NF-κB Family Antibody Sampler Kit II: Image 32

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

NF-κB Family Antibody Sampler Kit II: Image 33

Western blot analysis of extracts from various cell lines using RelB (D7D7W) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.

NF-κB Family Antibody Sampler Kit II: Image 34

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

To Purchase # 55764T
Product # Size Price
55764T
1 Kit  (6 x 20 µl) $ 445

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
NF-κB p65 (D14E12) XP® Rabbit mAb 8242 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
  • C&R
H M R Hm Mk Dg 65 Rabbit IgG
Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb 3033 20 µl
  • WB
  • IP
  • IF
  • F
H M R Hm Mk Pg 65 Rabbit IgG
RelB (D7D7W) Rabbit mAb 10544 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H 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 (D4P4D) Rabbit mAb 13586 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R 50 Active form. 120 Precursor Rabbit IgG
NF-κB2 p100/p52 (D7A9K) Rabbit mAb 37359 20 µl
  • WB
  • IP
  • ChIP
H 120, 52 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The NF-κB Family Antibody Sampler Kit II provides an economical means of detecting members of the NF-κB family. The kit includes enough antibody to perform two western blots with each primary antibody.

Specificity / Sensitivity

Each antibody in the kit detects endogenous levels of its intended target.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Glu498 of human NF-κB p65/RelA, Leu65 of human c-Rel, Ile415 of mouse NF-κB1 p105/p50, and residues near the amino terminus of human NF-κB2 p100/p52, a recombinant protein specific to the carboxy terminus of human RelB, and a synthetic phosphopeptide corresponding to residues surrounding Ser536 of human NF-κB p65/RelA.

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 U S A 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.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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