Revision 7

#14895Store 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, IP, IF-IC, FC-FP, ChIP, ChIP-seq, C&R

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

87-98

Source/Isotype:

Rabbit IgG

UniProt ID:

#P41182

Entrez-Gene Id:

604

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
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:200
Immunofluorescence (Immunocytochemistry) 1:50 - 1:100
Flow Cytometry (Fixed/Permeabilized) 1:800 - 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 #47899.

Specificity / Sensitivity

BCL6 (D4I2V) XP® Rabbit mAb recognizes endogenous levels of total BCL6 protein. It is predicted to detect both human BCL6 isoform 1 and isoform 2.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a recombinant protein fragment spanning residues Asp210 through Thr409 of human BCL6 protein.

Background

Chromosomal translocations result in misregulation of the proto-oncogene BCL6 in patients with B cell-derived non-Hodgkin's lymphoma (1). The BCL6 gene is selectively expressed in mature B cells and encodes a nuclear phosphoprotein that belongs to the BTB/POZ zinc finger family of transcription factors (2,3). BCL6 protein can bind to target DNA sequences of Stat6 and, analogous to Stat6, modulate the expression of interleukin-4-induced genes (4). Furthermore, BCL6 restrains p53-dependent senescence, making BCL6-active tumors functionally p53-negative (5). The mitogen-activated protein kinases, Erk1 and Erk2, but not JNK, phosphorylate BCL6 at multiple sites. Phosphorylation of BCL6 at Ser333 and Ser343 results in degradation of BCL6 by the ubiquitin/proteasome pathway in B cells (6,7). In addition, BCL6 is acetylated and its transcriptional repressor function is inhibited by the transcriptional co-activator p300 (8).

  1. Ye, B.H. et al. (1993) Science 262, 747-50.
  2. Onizuka, T. et al. (1995) Blood 86, 28-37.
  3. Zollman, S. et al. (1994) Proc Natl Acad Sci U S A 91, 10717-21.
  4. Harris, M.B. et al. (1999) Mol Cell Biol 19, 7264-75.
  5. Shvarts, A. et al. (2002) Genes Dev 16, 681-6.
  6. Moriyama, M. et al. (1997) Oncogene 14, 2465-74.
  7. Niu, H. et al. (1998) Genes Dev 12, 1953-61.
  8. Bereshchenko, O.R. et al. (2002) Nat Genet 32, 606-13.

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™ IP: Immunoprecipitation 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 7
#14895

BCL6 (D4I2V) XP® Rabbit mAb

Western Blotting Image 1: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
使用 BCL6 (D4I2V) XP® Rabbit mAb(上)和 β-Actin (D6A8) Rabbit mAb #8457(下),对不同细胞系提取物进行蛋白质印迹分析。
Western Blotting Image 1: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
使用 BCL6 (D4I2V) XP® Rabbit mAb #14895 对 Daudi 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)。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。
Immunoprecipitation Image 1: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
使用 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(泳道 2)或 BCL6 (D4I2V) XP® Rabbit mAb(泳道 3),对 Ramos 细胞提取物中的 BCL6 进行免疫沉淀。泳道 1 是 10% 输入对照。使用 BCL6 (D4I2V) XP® Rabbit mAb 进行蛋白质印迹分析。Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 用作二抗。
Immunofluorescence Image 1: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
使用 BCL6 (D4I2V) XP® Rabbit mAb(绿色),对 Ramos(左)和 Jurkat(右)细胞进行共聚焦免疫荧光分析。肌动蛋白微丝由 DyLight 554 Phalloidin #13054(红色)标记。
Flow Cytometry Image 1: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
使用 BCL6 (D4I2V) XP®3900 Rabbit mAb (实线)或浓度匹配的 (DA1E) mAb IgG XP® Isotype Control #(虚线)对 Jurkat 细胞(蓝色)和 Ramos 细胞(绿色)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: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
使用 SimpleChIP® 酶解染色质免疫共沉淀试剂盒(磁珠法) #9005 对 Raji 细胞的交联染色质和 BCL6 (D4I2V) XP® 兔单克隆抗体进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在 BCL6 的已知靶基因 HSP90AB1/HSP90β 内结合(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 2: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
使用 SimpleChIP® 酶解染色质免疫共沉淀试剂盒(磁珠法) #9005 对 Raji 细胞的交联染色质和 BCL6 (D4I2V) XP® 兔单克隆抗体进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在染色体 6(上图)内的结合,包括 BCL6 的已知靶标基因 HSP90AB1/HSP90β(下图)(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 3: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对 Raji 细胞中提取的交联染色质,在加入 BCL6 (D4I2V) XP® Rabbit mAb 或 Normal Rabbit IgG #2729 后进行染色质免疫沉淀。使用 SimpleChIP® Human HSP90β Promoter Primers #12899 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: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652,以 Raji 细胞和 BCL6 (D4I2V) 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 BCL6 的已知靶标基因 GSK3A 基因(参见包含 CUT&RUN-qPCR 数据的其他结果图)。
CUT and RUN Image 2: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652,以 Raji 细胞和 BCL6 (D4I2V) 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。这些图显示结合作用遍及第 19 号染色体(上图),包括 BCL6 的已知靶标基因 GSK3A(下图)(参见包含 CUT&RUN-qPCR 数据的其他结果图)。
CUT and RUN Image 3: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 ,以 Raji 细胞和 BCL6 (D4I2V) XP® Rabbit mAb 或 Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 进行 CUT&RUN。使用人 GSK3A 启动子引物、人 GNB2 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。