Revision 7

#14895Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) orders@cellsignal.com

Support: 877-678-TECH (8324)

Web: info@cellsignal.com 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.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 7
#14895

BCL6 (D4I2V) XP® Rabbit mAb

Western Blotting Image 1: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using BCL6 (D4I2V) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 1: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
Simple Western™ analysis of lysates (0.1 mg/mL) from Daudi cells using BCL6 (D4I2V) XP® Rabbit mAb #14895. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Immunoprecipitation Image 1: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
Immunoprecipitation of BCL6 from Ramos cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or BCL6 (D4I2V) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using BCL6 (D4I2V) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody.
Immunofluorescence Image 1: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
Confocal immunofluorescent analysis of Ramos (left) and Jurkat (right) cells using BCL6 (D4I2V) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red).
Flow Cytometry Image 1: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
Flow cytometric analysis of Jurkat cells (blue) and Ramos cells (green) using BCL6 (D4I2V) XP® Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from Raji cells and BCL6 (D4I2V) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across HSP90AB1/HSP90β, a known target gene of BCL6 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from Raji cells and BCL6 (D4I2V) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 6 (upper), including HSP90AB1/HSP90β (lower), a known target gene of BCL6 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from Raji cells and either BCL6 (D4I2V) XP® Rabbit mAb or Normal Rabbit IgG #2729, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human HSP90β Promoter Primers #12899 and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT and RUN Image 1: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
CUT&RUN was performed with Raji cells and BCL6 (D4I2V) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across GSK3A gene, a known target gene of BCL6 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
CUT&RUN was performed with Raji cells and BCL6 (D4I2V) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 19 (upper), including GSK3A (lower), a known target gene of BCL6 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: BCL6 (D4I2V) XP® Rabbit mAb Expand Image
CUT&RUN was performed with Raji cells and either BCL6 (D4I2V) XP® Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human GSK3A promoter primers, human GNB2 promoter primers, and human OCM2 exon 1 primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.