Revision 5

#4764Store at -20C

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.
Applications:

WB, IF-IC, FC-FP

REACTIVITY:

H M R Mk B

SENSITIVITY:

Endogenous

MW (kDa):

65

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q04206

Entrez-Gene Id:

5970

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunofluorescence (Immunocytochemistry) 1:50 - 1:200
Flow Cytometry (Fixed/Permeabilized) 1:400

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 #66286.

Specificity / Sensitivity

NF-κB p65 (C22B4) Rabbit mAb detects endogenous levels of total NF-κB p65 protein. This antibody may also cross-react with the p50 subunit.

Species Reactivity:

Human, Mouse, Rat, Monkey, Bovine

Species predicted to react based on 100% sequence homology

Dog

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human NF-κB p65.

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.

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 BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized)

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.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

除非 CST 的合法授书代表以书面形式书行明确同意,否书以下条款适用于 CST、其关书方或分书商提供的书品。 任何书充本条款或与本条款不同的客书条款和条件,除非书 CST 的合法授书代表以书面形式书独接受, 否书均被拒书,并且无效。

专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品, (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Revision 5
#4764

NF-κB p65 (C22B4) Rabbit mAb

Western Blotting Image 1: NF-κB p65 (C22B4) Rabbit mAb Expand Image
使用 NF-κB p65 (C22B4) Rabbit mAb #4764 (上图)或 β-actin (13E5) Rabbit mAb #4970 (下图)对对照 HeLa 细胞(泳道 1) 或 NF-κB p65 敲除型 HeLa 细胞(泳道 2)的提取物进行蛋白质印迹法分析。NF-κB p65 敲除型 HeLa 细胞中没有信号,这证实了该抗体对 NF-κB p65 的特异性。
Western Blotting Image 2: NF-κB p65 (C22B4) Rabbit mAb Expand Image
使用 Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033(上)和 NF-κB p65 (C22B4) Rabbit mAb(下)时,对来自 THP-1 细胞(用 TPA(#9905,80 nM 24 小时)分化并用 1 μg/ml LPS 处理所示时间)的提取物进行蛋白质印迹分析。
Western Blotting Image 3: NF-κB p65 (C22B4) Rabbit mAb Expand Image
使用 NF-κB p65 (C22B4) Rabbit mAb 对来自 HeLa 细胞(人)、PC12 细胞(大鼠)和 Neuro2A 细胞(小鼠)细胞系的提取物进行蛋白质印迹分析。
Western Blotting Image 4: NF-κB p65 (C22B4) Rabbit mAb Expand Image
使用 NF-κB p65 (C22B4) Rabbit mAb #4764 和 α-Tubulin (11H10) Rabbit mAb #2125 ,对转染 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-)、SignalSilence® NF-κB p65 siRNA II (+) 或 SignalSilence® NF-κB p65 siRNA I #6261 (+) 的 HeLa 细胞的提取物进行蛋白质印迹分析。NF-κB p65 (C22B4) Rabbit mAb 证实 NF-κB p65 沉默并且 α-Tubulin (11H10) Rabbit mAb 用来作为 NF-κB p65 siRNA 的载量和特异性的对照。
Immunofluorescence Image 1: NF-κB p65 (C22B4) Rabbit mAb Expand Image
使用 NF-κB p65 (C22B4) Rabbit mAb(绿色)对 HeLa 细胞(未处理(左)或 TNF-α 处理(#8902,20 ng/ml 20 分钟,右))进行共聚焦免疫荧光分析。肌动蛋白纤丝用 Alexa Fluor®555 phalloidin(红色)进行标记。蓝色伪彩 = DRAQ5 (DNA 荧光染料)。
Flow Cytometry Image 1: NF-κB p65 (C22B4) Rabbit mAb Expand Image
使用 NF-κB p65 (C22B4) Rabbit mAb(实线)或浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)对 HeLa 细胞进行流式细胞分析。Anti-rabbit IgG (H+L)、F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 作为二抗。