Revision 18

#8242

Store at -20C

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Applications:
W, W-S, IP, IHC-P, IF-F, IF-IC, FC-FP, ChIP, ChIP-seq, C&R

Reactivity:
H M R Hm Mk Dg

Sensitivity:
Endogenous

MW (kDa):
65

Source/Isotype:
Rabbit IgG

UniProt ID:
#Q04206

Entrez-Gene Id:
5970

Product Usage Information

For optimal ChIP and ChIP-seq results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:100
Immunohistochemistry (Paraffin) 1:400 - 1:1600
Immunofluorescence (Frozen) 1:400
Immunofluorescence (Immunocytochemistry) 1:400 - 1:800
Flow Cytometry (Fixed/Permeabilized) 1:400 - 1:1600
Chromatin IP 1:100
Chromatin IP-seq 1:100
CUT&RUN 1:100

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.

For a carrier free (BSA and azide free) version of this product see product #69994.

Specificity/Sensitivity

NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody recognizes endogenous levels of total NF-κB p65/RelA protein. This antibody does not cross react with other NF-κB/Rel family members. Non-specific staining was observed in pancreatic islets by immunohistochemistry.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Glu498 of human NF-κB p65/RelA protein.

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).

Species Reactivity

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

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

W: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-F: Immunofluorescence (Frozen) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN

Cross-Reactivity Key

H: Human M: Mouse R: Rat Hm: Hamster Mk: Monkey Dg: Dog

Trademarks and Patents

Cell Signaling Technology is a 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

使用に関する制限

法的な権限を与えられたCSTの担当者が署名した書面によって別途明示的に合意された場合を除き、CST、その関連会社または代理店が提供する製品には以下の条件が適用されます。お客様が定める条件でここに定められた条件に含まれるものを超えるもの、または、ここに定められた条件と異なるものは、法的な権限を与えられたCSTの担当者が別途書面にて受諾した場合を除き、拒絶され、いかなる効力も効果も有しません。

研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 18

Cell Signaling Technologyロゴ
Western blot analysis of extracts from various cell lines using NF-κB p65 (D14E12) XP® Rabbit mAb.
Western Blotting Image 1: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Simple Western™ analysis of lysates (1 mg/mL) from HeLa cells using NF-κB p65 (D14E12) XP® Rabbit mAb #8242. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Western Blotting Image 1: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Immunoprecipitation of NF-kB p65 from CHO cell extracts. Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is NF-κB p65 (D14E12) XP® Rabbit mAb, #8242. Western blot was performed using NF-κB p65 (L8F6) Mouse mAb, #6956.
Immunoprecipitation Image 1: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 18

Cell Signaling Technologyロゴ
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 (right)).
Immunohistochemistry Image 1: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Immunohistochemical analysis of paraffin-embedded human chronic cholecystitis using NF-κB p65 (D14E12) XP® Rabbit mAb.
Immunohistochemistry Image 2: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using NF-κB p65 (D14E12) XP® Rabbit mAb.
Immunohistochemistry Image 3: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 18

Cell Signaling Technologyロゴ
Immunohistochemical analysis of paraffin-embedded normal human small intestine using NF-κB p65 (D14E12) XP® Rabbit mAb.
Immunohistochemistry Image 4: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Immunohistochemical analysis of paraffin-embedded human gastric carcinoma using NF-κB p65 (D14E12) XP® Rabbit mAb.
Immunohistochemistry Image 5: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Immunohistochemical analysis of paraffin-embedded human thymoma using NF-κB p65 (D14E12) XP® Rabbit mAb.
Immunohistochemistry Image 6: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 18

Cell Signaling Technologyロゴ
Immunohistochemical analysis of paraffin-embedded human endometrioid carcinoma using NF-κB p65 (D14E12) XP® Rabbit mAb.
Immunohistochemistry Image 7: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Immunohistochemical analysis of paraffin-embedded human adenoid cystic carcinoma of the salivary gland (left), squamous cell lung carcinoma (middle) or normal colon (right) using NF-κB p65 (D14E12) XP® Rabbit mAb (top) or an
NF-κB p65 Mouse mAb (bottom). These two antibodies detect unique, non-overlapping epitopes on human NF-κB p65. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.
Immunohistochemistry Image 8: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Confocal immunofluorescent analysis of fixed frozen kidney from untreated (left) or LPS-treated mice (2 hrs, 5 mg/kg) (right) using NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody (green) and Lamin A/C (4C11) Mouse Monoclonal Antibody (Alexa Fluor® 647 Conjugate) #41357 (red).
Immunofluorescence Image 1: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 18

Cell Signaling Technologyロゴ
Confocal immunofluorescent analysis of HT-1080 cells, untreated (left) or treated with hTNF-alpha (20 ng/ml, 20 min; right), using NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody (green), DyLight® 554 Phalloidin #13054 (red), and DRAQ5® #4084 (blue).
Immunofluorescence Image 1: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Flow cytometric analysis of fixed and permeabilized Jurkat cells using NF-κB p65 (D14E12) Rabbit Monoclonal Antibody (solid line) or concentration-matched Rabbit (DA1E) Monoclonal Antibody IgG Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Flow Cytometry Image 1: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α (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 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #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 datasheet.
Chromatin Immunoprecipitation Image 1: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 18

Cell Signaling Technologyロゴ
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α (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 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 4 (upper), including IL-8 (lower), a known target gene of NFκB (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α (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.
Chromatin Immunoprecipitation Image 3: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
CUT&RUN was performed with HeLa cells treated with hTNF-α (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across LAMC2, a known target gene of NF-κB p65 (see additional figure containing CUT&RUN-qPCR data).
CUT & RUN Image 1: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 18

Cell Signaling Technologyロゴ
CUT&RUN was performed with HeLa cells treated with hTNF-α (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #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).
CUT & RUN Image 2: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
CUT&RUN was performed with HeLa cells treated with hTNF-α (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.
CUT & RUN Image 3: NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.