Revision 8
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, IP, IHC-P, IF-IC, FC-FP, ChIP, ChIP-seq, C&R

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

90 (PR-A), 118 (PR-B)

Source/Isotype:

Rabbit IgG

UniProt ID:

#P06401

Entrez-Gene Id:

5241

Product Information

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
Immunoprecipitation 1:50
Immunohistochemistry (Paraffin) 1:500 - 1:2000
Immunofluorescence (Immunocytochemistry) 1:800 - 1:1600
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 #18444.

Specificity / Sensitivity

Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb recognizes endogenous levels of total progesterone receptor A and B proteins. This antibody does not cross-react with either the glucocorticoid receptor or the mineralocorticoid receptor.

Species Reactivity:

Human

Species predicted to react based on 100% sequence homology

Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr541 of human progesterone receptor protein.

Background

Human progesterone receptor (PR) is expressed as two forms: the full length PR-B and the short form PR-A. PR-A lacks the first 164 amino acid residues of PR-B (1,2). Both PR-A and PR-B are ligand activated, but differ in their relative ability to activate target gene transcription (3,4). The activity of PR is regulated by phosphorylation; at least seven serine residues are phosphorylated in its amino-terminal domain. Three sites (Ser81, Ser102, and Ser162) are unique to full length PR-B, while other sites (Ser190, Ser294, Ser345, and Ser400) are shared by both isoforms (5). Phosphorylation of PR-B at Ser190 (equivalent to Ser26 of PR-A) is catalyzed by CDK2 (6). Mutation of Ser190 results in decreased activity of PR (7), suggesting that the phosphorylation at Ser190 may be critical to its biological function.

  1. Evans, R.M. (1988) Science 240, 889-895.
  2. Kastner, P. et al. (1990) EMBO J. 112, 1603-1614.
  3. Giangrande, P.H. et al. (2000) Mol. Cell. Biol. 20, 3102-3115.
  4. Wen, D.X. et al. (1994) Mol. Cell. Biol. 14, 8356-8364.
  5. Clemm, D.L. et al. (2000) Mol. Endocrinol. 14, 52-65.
  6. Zhang, Y. et al. (1997) Mol. Endocrinol. 11, 823-832.
  7. Takimoto, G.S. et al. (1996) J. Biol. Chem. 271, 13308-13316.

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 IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) 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.
XP is a registered trademark of Cell Signaling Technology, Inc.
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 8
#8757

Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb

Western Blotting Image 1: Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb Expand Image
使用 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb(上图)或 GAPDH (D16H11) XP® Rabbit mAb #5174(下图),对 T-47D(PR 阳性)和 MDA-MB-231(PR 阴性)细胞的提取物进行蛋白质印迹分析。
Western Blotting Image 2: Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb Expand Image
使用 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb(上图)或 GAPDH (D16H11) XP® Rabbit mAb #5174(下图),对在添加了 5% 活性炭剥离 FBS 的无酚红培养基中生长 48 小时,然后经赋形剂对照物 (-) 或普美孕酮(R5020,100 nM,16小时;+)处理的 T-47D 细胞的提取物进行蛋白质印迹分析。用 R5020 长时间处理表达 PR 的细胞已知会诱导 PR 表达下调和过度磷酸化,这表现为在 SDS-PAGE 上的迁移变慢。
No image available
Immunohistochemistry Image 1: Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb Expand Image
使用 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb 对石蜡包埋的人乳腺浸润性导管癌细胞进行免疫组织化学分析。
Immunohistochemistry Image 2: Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb Expand Image
使用 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb 对石蜡包埋的 T-47D (高 PR,左图)、MCF-7 (低 PR,中间)和 MDA-MB-231 (PR 阴性,右图)细胞沉淀物进行免疫组织化学分析。
Immunofluorescence Image 1: Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb Expand Image
使用 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb(绿色),对 T-47D(PR 阳性,左图)和 MDA-MB-231 (PR 阴性,右图)细胞进行共聚焦免疫荧光分析。使用 DY-554 phalloidin(红色)标记肌动蛋白纤丝。
Flow Cytometry Image 1: Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb Expand Image
使用 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb(实线)或浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)对 MDA-MB-231 细胞(蓝色,阴性)和 T47D 细胞(绿色,阳性)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: Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对在含有 5% 活性炭剥离 FBS 的培养基中培养 48 小时,然后经普美孕酮(R5020, 10 nM, 1 小时)处理的 T-47D 细胞中提取的染色质,在加入 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb 后,进行染色质免疫沉淀。DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795。结果图显示在 FKBP5/FKBP51 内结合,FKBP5/FKBP51 是 Progesterone Receptor A/B 的一个已知靶标基因(见其他含 ChIP-qPCR 数据的图)。欲知其他 ChIP-seq 情况,请下载产品说明书。
Chromatin Immunoprecipitation Image 2: Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对在含有 5% 活性炭剥离 FBS 的培养基中培养 48 小时,然后经普美孕酮(R5020, 10 nM, 1 小时)处理的 T-47D 细胞中提取的染色质,在加入 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb 后,进行染色质免疫沉淀分析。DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795。结果图显示了染色体 6(上图)内的结合,包括雄激素受体 A/B 的已知靶基因 FKBP5/ FKBP51(下图)(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 3: Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb Expand Image
T-47D 细胞在添加了 5% 活性炭剥离 FBS 的无酚红培养基中培养 48 小时,然后不进行处理(左小图)或用普美孕酮处理(R5020,10 nM,1 小时;右小图)。使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对细胞的交联染色质与 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb 或Normal Rabbit IgG #2729 进行染色质免疫沉淀。使用 SimpleChIP® Human FKBP51 Intron 5 Primers #7859、human E2F-1 proximal enhancer site #1 primers 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: Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652,以在含 5% 活性炭剥离的 FBS 的无酚红培养基中培养 48 小时、然后用孕激素(R5020,10 nM,1 小时)处理的 T-47D 细胞和 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb 进行 CUT&RUN。使用 DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。该图显示结合作用遍及 SETD9 基因。
CUT and RUN Image 2: Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652,以在含 5% 活性炭剥离的 FBS 的无酚红培养基中培养 48 小时、然后用孕激素(R5020,10 nM,1 小时)处理的 T-47D 细胞和 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb 进行 CUT&RUN。使用 DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。该图显示结合作用遍及染色体 5(上图),包括 SETD9(下图)。
CUT and RUN Image 3: Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652,以在含 5% 活性炭剥离的 FBS 的无酚红培养基中培养 48 小时、然后用孕激素(R5020,10 nM,1 小时)处理的 T-47D 细胞和 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb 或 Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 进行 CUT&RUN。使用人 TBL2 启动子引物和人 OCM2 外显子 1 引物,通过实时 PCR 对富集的 DNA 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。