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.

使用に関する制限

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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.