Revision 9

#2250Store 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, W-S, IF-F, IF-IC, FC-FP, ChIP

REACTIVITY:

H M R

SENSITIVITY:

Endogenous

MW (kDa):

62

Source/Isotype:

Rabbit IgG

UniProt ID:

#P01100

Entrez-Gene Id:

2353

Product Information

Product Usage Information

For optimal ChIP results, use 10 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.

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunofluorescence (Frozen) 1:1600 - 1:3200
Immunofluorescence (Immunocytochemistry) 1:3200 - 1:12800
Flow Cytometry (Fixed/Permeabilized) 1:1600 - 1:6400
Chromatin IP 1:50

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 #53345.

Specificity / Sensitivity

This antibody detects endogenous levels of total c-Fos protein. The antibody does not cross-react with other Fos proteins, including FosB, FRA1 and FRA2. c-Fos (9F6) Rabbit mAb #2250 non-specifically stains fixed frozen mouse spleen and liver by immunofluorescence.

Species Reactivity:

Human, Mouse, Rat

Species predicted to react based on 100% sequence homology

Hamster, Bovine, Pig

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human c-Fos protein.

Background

The Fos family of nuclear oncogenes includes c-Fos, FosB, Fos-related antigen 1 (FRA1), and Fos-related antigen 2 (FRA2) (1). While most Fos proteins exist as a single isoform, the FosB protein exists as two isoforms: full-length FosB and a shorter form, FosB2 (Delta FosB), which lacks the carboxy-terminal 101 amino acids (1-3). The expression of Fos proteins is rapidly and transiently induced by a variety of extracellular stimuli, including growth factors, cytokines, neurotransmitters, polypeptide hormones, and stress. Fos proteins dimerize with Jun proteins (c-Jun, JunB, and JunD) to form Activator Protein-1 (AP-1), a transcription factor that binds to TRE/AP-1 elements and activates transcription. Fos and Jun proteins contain the leucine-zipper motif that mediates dimerization and an adjacent basic domain that binds to DNA. The various Fos/Jun heterodimers differ in their ability to transactivate AP-1 dependent genes. In addition to increased expression, phosphorylation of Fos proteins by Erk kinases in response to extracellular stimuli may further increase transcriptional activity (4-6). Phosphorylation of c-Fos at Ser32 and Thr232 by Erk5 increases protein stability and nuclear localization (5). Phosphorylation of FRA1 at Ser252 and Ser265 by Erk1/2 increases protein stability and leads to overexpression of FRA1 in cancer cells (6). Following growth factor stimulation, expression of FosB and c-Fos in quiescent fibroblasts is immediate, but very short-lived, with protein levels dissipating after several hours (7). FRA1 and FRA2 expression persists longer, and appreciable levels can be detected in asynchronously growing cells (8). Deregulated expression of c-Fos, FosB, or FRA2 can result in neoplastic cellular transformation; however, Delta FosB lacks the ability to transform cells (2,3).

  1. Tulchinsky, E. (2000) Histol Histopathol 15, 921-8.
  2. Dobrazanski, P. et al. (1991) Mol Cell Biol 11, 5470-8.
  3. Nakabeppu, Y. and Nathans, D. (1991) Cell 64, 751-9.
  4. Rosenberger, S.F. et al. (1999) J Biol Chem 274, 1124-30.
  5. Sasaki, T. et al. (2006) Mol Cell 24, 63-75.
  6. Basbous, J. et al. (2007) Mol Cell Biol 27, 3936-50.
  7. Kovary, K. and Bravo, R. (1991) Mol Cell Biol 11, 2451-9.
  8. Kovary, K. and Bravo, R. (1992) Mol Cell Biol 12, 5015-23.

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

WB: Western Blotting W-S: Simple Western™ IF-F: Immunofluorescence (Frozen) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP

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.
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 9
#2250

c-Fos (9F6) Rabbit mAb

Western Blotting Image 1: c-Fos (9F6) Rabbit mAb Expand Image
使用 c-Fos (9F6) Rabbit mAb 对 HeLa 细胞和 H-4-II-E 细胞(血清饥饿过夜和 TPA 刺激 4小时)的提取物进行蛋白质印迹分析。
Western Blotting Image 2: c-Fos (9F6) Rabbit mAb Expand Image
使用 c-Fos (9F6) Rabbit mAb #2250(上图)和 β-Actin (13E5) Rabbit mAb (HRP Conjugate) #5125(下图)对血清饥饿过夜且未经处理然后经过 TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174(泳道 2)刺激 4 小时或经过 TPA 刺激 4 小时再然后经过 λ-磷酸酶处理的 NIH/3T3 细胞提取物进行蛋白质印迹分析。
Western Blotting Image 1: c-Fos (9F6) Rabbit mAb Expand Image
使用 c-Fos (9F6) Rabbit mAb #2250 对经过 TPA(400 nM,4 小时)处理的血清饥饿 HeLa 细胞的裂解物 (1.0 mg/mL) 进行 Simple Western™ 分析。虚拟泳道式图像(左图)显示一抗稀释比例为 1:10 和 1:50 时的靶标条带(如图所示)。对应的电泳图(右图)为一抗稀释比例在 1:10(蓝线)和 1:50(绿线)时沿毛细血管内分子量的化学发光结果。在还原条件下,使用 12-230 kDa 分离模块在 ProteinSimple(BioTechne 品牌)的 Jess™ Simple Western 仪器上进行该实验。
Immunofluorescence Image 1: c-Fos (9F6) Rabbit mAb Expand Image
使用 c-Fos (9F6) Rabbit mAb #2250(绿色)和 Neurofilament-L (DA2) Mouse mAb #2835(红色)对小鼠大脑皮层进行共聚焦免疫荧光分析。样本用 ProLong® Gold Antifade Reagent with DAPI #8961(蓝色)封片。
Immunofluorescence Image 2: c-Fos (9F6) Rabbit mAb Expand Image
使用 c-Fos (9F6) Rabbit mAb #2250(绿色)和 Neurofilament-L (DA2) Mouse mAb #2835(红色)进行小鼠标桥进行共聚焦免疫荧光分析。样本用 ProLong® Gold Antifade Reagent with DAPI #8961(蓝色)封片。
Immunofluorescence Image 1: c-Fos (9F6) Rabbit mAb Expand Image
对血清饥饿(左)或用 TPA (#9905) 处理 4小时的(右)、并用 c-Fos (9F6) Rabbit mAb (绿色)标签的 HeLa 细胞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。
Flow Cytometry Image 1: c-Fos (9F6) Rabbit mAb Expand Image
使用 c-Fos (9F6) Rabbit mAb(实线)或浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线),对未经处理(蓝色,中度表达)或已经过 TSA #4174(200 μM,4小时;绿色,高度表达)处理的饥饿过夜的 HeLa 细胞血清进行流式细胞分析。Anti-rabbit IgG (H+L)、F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 作为二抗。
Chromatin Immunoprecipitation Image 1: c-Fos (9F6) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对饥饿过夜并用 Human β-Nerve Growth Factor (h-βNGF) #5221(50ng/ml) 处理 2 小时)的 PC-12 细胞中提取的交联染色质,在加入 c-Fos (9F6) Rabbit mAb 或 Normal Rabbit IgG #2729 后,进行染色质免疫沉淀。使用 SimpleChIP® Rat CCRN4L Promoter Primers #7983、rat DCLK1 promoter primers 和 SimpleChIP® Rat GAPDH Promoter Primers #7964,通过实时 PCR 对富集的 DNA 进行定量。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。