Revision 9
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, IHC-P, IF-F, IF-IC, FC-FP, ChIP, ChIP-seq, C&R

REACTIVITY:

H M R

SENSITIVITY:

Endogenous

MW (kDa):

43

Source/Isotype:

Rabbit IgG

UniProt ID:

#P16220

Entrez-Gene Id:

1385

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq 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.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunohistochemistry (Paraffin) 1:400 - 1:1600
Immunofluorescence (Frozen) 1:400 - 1:1600
Immunofluorescence (Immunocytochemistry) 1:400 - 1:1600
Flow Cytometry (Fixed/Permeabilized) 1:400 - 1:1600
Chromatin IP 1:50
Chromatin IP-seq 1:50
CUT&RUN 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 #39561.

Specificity / Sensitivity

Phospho-CREB (Ser133) (87G3) Rabbit mAb detects endogenous levels of CREB only when phosphorylated at serine 133. The antibody also detects the phosphorylated form of the CREB-related protein, ATF-1.

Species Reactivity:

Human, Mouse, Rat

Species predicted to react based on 100% sequence homology

Zebrafish

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser133 of human CREB.

Background

CREB is a bZIP transcription factor that activates target genes through cAMP response elements. CREB is able to mediate signals from numerous physiological stimuli, resulting in regulation of a broad array of cellular responses. While CREB is expressed in numerous tissues, it plays a large regulatory role in the nervous system. CREB is believed to play a key role in promoting neuronal survival, precursor proliferation, neurite outgrowth, and neuronal differentiation in certain neuronal populations (1-3). Additionally, CREB signaling is involved in learning and memory in several organisms (4-6). CREB is able to selectively activate numerous downstream genes through interactions with different dimerization partners. CREB is activated by phosphorylation at Ser133 by various signaling pathways, including Erk, Ca2+, and stress signaling. Some of the kinases involved in phosphorylating CREB at Ser133 are p90RSK, MSK, CaMKIV, and MAPKAPK-2 (7-9).

  1. Lonze, B.E. et al. (2002) Neuron 34, 371-85.
  2. Lee, M.M. et al. (1999) J Neurosci Res 55, 702-12.
  3. Redmond, L. et al. (2002) Neuron 34, 999-1010.
  4. Dash, P.K. et al. (1990) Nature 345, 718-21.
  5. Yin, J.C. et al. (1994) Cell 79, 49-58.
  6. Guzowski, J.F. and McGaugh, J.L. (1997) Proc Natl Acad Sci USA 94, 2693-8.
  7. Xing, J. et al. (1998) Mol Cell Biol 18, 1946-55.
  8. Ribar, T.J. et al. (2000) J Neurosci 20, RC107.
  9. Tan, Y. et al. (1996) EMBO J 15, 4629-42.

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 W-S: Simple Western™ 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 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
#9198

Phospho-CREB (Ser133) (87G3) Rabbit mAb

Western Blotting Image 1: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用 Phospho-CREB (Ser133) (87G3) Rabbit mAb(上)或 CREB (48H2) Rabbit mAb #9197 (下),对未处理或经毛喉素处理和 FGF 处理的 SK-N-MC 细胞提取物进行蛋白质印迹分析。
Western Blotting Image 1: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用 Phospho-CREB (Ser133) (87G3) Rabbit mAb #9198 对经毛喉素处理(30uM,30 分钟)和 IBMX处理(0.5mM,30 分钟)的 SK-N-MC 细胞的裂解物 (0.1 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)。该抗体也检出磷酸化形式的 CREB 相关蛋白——ATF-1 (如所示)。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。使用 12-230 kDa 分离模块在 ProteinSimple(BioTechne 品牌)的 Jess™ Simple Western 仪器上还原条件下进行该实验。
Immunohistochemistry Image 1: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用 Phospho-CREB (Ser133) (87G3) Rabbit mAb,对石蜡包埋的人肺癌进行免疫组织化学分析,并显示胞核染色。
Immunohistochemistry Image 2: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用 Phospho-CREB (Ser133) (87G3) Rabbit mAb,对石蜡包埋的小鼠肺进行免疫组织化学分析。
Immunohistochemistry Image 3: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
在对照肽(左)或 Phospho-CREB (Ser133) Blocking Peptide #1090(右)存在的情况下,使用 Phospho-CREB (Ser133) (87G3) Rabbit mAb,对石蜡包埋的人乳腺癌进行免疫组织化学分析。
Immunohistochemistry Image 4: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用 Phospho-CREB (Ser133) (87G3) Rabbit mAb,对未处理(左)或经 IBMX 及毛喉素处理(右)的石蜡包埋的 SK-N-MC 细胞进行的免疫组织化学分析,显示诱导的胞核着染。
Immunohistochemistry Image 5: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用 Phospho-CREB (Ser133) (87G3) Rabbit mAb,对未处理(左)或 λ 磷酸酶处理(右)的石蜡包埋的人肾细胞癌进行免疫组织化学分析。
Immunofluorescence Image 1: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
对用 Phospho-CREB (Ser133) (87G3) Rabbit mAb(红色)、Neurofilament-L (DA2) Mouse mAb #2835(蓝色)和 Phospho-S6 Ribosomal Protein (Ser235/236) (2F9) Rabbit mAb (Alexa Fluor® 488 Conjugate) #4854 标记的接受假手术(左图)或让之缺血 15 分钟随后再灌注 30 分钟(中图)和 4 小时(右图)的大鼠齿状回细胞进行共聚焦免疫荧光图像分析。
Immunofluorescence Image 1: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
与未处理细胞(右)相比,使用 Phospho-CREB (Ser133) (87G3) Rabbit mAb(左,红),对 Forskolin 和 IBMX 处理 25 分钟后显示核染色的 SK-N-MC 细胞进行共聚焦显微镜图像。蓝色伪彩 = DRAQ5® #4084(DNA 荧光染料)。
Flow Cytometry Image 1: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用 Phospho-CREB (Ser133) (87G3) Rabbit mAb(实线)或浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)对未经过处理(蓝色)或经过 TPA #4174 (500nM) 和碘霉素#9995(1 µM,4 小时;绿色)处理的 Jurkat 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。
Chromatin Immunoprecipitation Image 1: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 对经 Forskolin #3828 (30 μM) 处理 1 小时的 293 细胞的交联染色质与 Phospho-CREB (Ser133) (87G3) Rabbit mAb 或 CREB (48H2) Rabbit mAb (#9197) 进行染色质免疫沉淀。使用 SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示与 NR4A3 结合,NR4A3 是磷酸 CREB 和 CREB 的一个已知靶标基因(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 2: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 对经 Forskolin #3828 (30 μM) 处理 1 小时的 293 细胞的交联染色质与 Phospho-CREB (Ser133) (87G3) Rabbit mAb 或 CREB (48H2) Rabbit mAb (#9197) 进行染色质免疫沉淀。使用 SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在染色体9(上图)内的结合,包括 Phospho-CREB 和 CREB 的已知靶标基因 NR4A3 (下图)(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 3: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 对经 Forskolin #3828 (30 μM) 处理 1 小时的 293 细胞的交联染色质与 Phospho-CREB (Ser133) (87G3) Rabbit mAb 或 Normal Rabbit IgG #2729 进行染色质免疫沉淀。使用人 ALS2 外显子 1 引物、SimpleChIP® Human NR4A3 Promoter Primers #4829 和 SimpleChIP® Human α Satellite Repeat Primers #4486,通过实时 PCR 对富集的 DNA 进行定量分析。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。
CUT and RUN Image 1: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用经 Forskolin # 3828 (30 μm) 处理 1h 的 293 细胞和 Phospho-CREB (Ser133) (87G3) Rabbit Mab,通过 CUT&RUN Assay Kit #86652进行 CUT&RUN。使用 SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在 NR4A3 基因内结合。
CUT and RUN Image 2: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用经 Forskolin # 3828 (30 μm) 处理 1h 的 293 细胞和 Phospho-CREB (Ser133) (87G3) Rabbit Mab,通过 CUT&RUN Assay Kit #86652进行 CUT&RUN。使用 SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-Seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在 9号染色体(上图)内的结合,包括 NR4A3 基因(下图)。
CUT and RUN Image 3: Phospho-CREB (Ser133) (87G3) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652,通过用 Forskolin #3828 (30 μM) 处理 1h 的293 细胞和 Phospho-CREB (Ser133) (87G3) Rabbit mAb 或 Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362,进行CUT&RUN。使用人源ALS2 外显子 1 引物和 SimpleChIP® Human α Satellite Repeat Primers #4486,通过实时 PCR 对富集的 DNA 进行定量分析。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。