Revision 6

#36426Store 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, IP, FC-FP, ChIP, C&R

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

58, 63-70, 85

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q9UKS7

Entrez-Gene Id:

22807

Product Information

Product Usage Information

For optimal ChIP 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
Immunoprecipitation 1:200
Flow Cytometry (Fixed/Permeabilized) 1:100
Chromatin IP 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 #54887.

Specificity / Sensitivity

Helios (D4Z6D) Rabbit mAb recognizes endogenous levels of total Helios protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro424 of human Helios protein.

Background

Helios (Ikaros family zinc finger 2, IKZF2) is an Ikaros family transcription factor composed of several zinc fingers that mediate DNA binding and homodimerization or heterodimerization with other Ikaros family proteins (1,2). In the hematopoietic system, Helios expression is restricted to T cells and early hematopoietic progenitors (1,2). In regulatory T cells, expression of Helios contributes to an anergic phenotype by binding to the IL-2 promoter and suppressing IL-2 transcription (3). In addition, alteration of the corresponding Helios gene IKZF2 is one hallmark of low-hypodiploid acute lymphoblastic leukemia (4).

  1. Hahm, K. et al. (1998) Genes Dev 12, 782-96.
  2. Kelley, C.M. et al. (1998) Curr Biol 8, 508-15.
  3. Baine, I. et al. (2013) J Immunol 190, 1008-16.
  4. Holmfeldt, L. et al. (2013) Nat Genet 45, 242-52.

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 IP: Immunoprecipitation FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP 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 6
#36426

Helios (D4Z6D) Rabbit mAb

Western Blotting Image 1: Helios (D4Z6D) Rabbit mAb Expand Image
Western blot analysis of extracts from 293T cells, untransfected (-) or transfected with a construct expressing Myc-tagged full-length human Helios (hHelios-Myc; +), using Helios (D4Z6D) Rabbit mAb.
Western Blotting Image 2: Helios (D4Z6D) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using Helios (D4Z6D) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunoprecipitation Image 1: Helios (D4Z6D) Rabbit mAb Expand Image
Immunoprecipitation of Helios from Jurkat cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Helios (D4Z6D) Rabbit mAb. Western blot analysis was performed using Helios (D4Z6D) Rabbit mAb.
Flow Cytometry Image 1: Helios (D4Z6D) Rabbit mAb Expand Image
Flow cytometric analysis of RL (blue) and Jurkat (green) cells using Helios (D4Z6D) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary anitbody.
Chromatin Immunoprecipitation Image 1: Helios (D4Z6D) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from Jurkat cells and either Helios (D4Z6D) 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 IKZF3 Promoter Primers #99663, human IL-2 promoter primers, 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: Helios (D4Z6D) Rabbit mAb Expand Image
CUT&RUN was performed with Jurkat cells and either Helios (D4Z6D) Rabbit mAb or Ikaros (D10E5) Rabbit mAb #9034, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across DARS2 gene.
CUT and RUN Image 2: Helios (D4Z6D) Rabbit mAb Expand Image
CUT&RUN was performed with Jurkat cells and either Helios (D4Z6D) Rabbit mAb or Ikaros (D10E5) Rabbit mAb #9034, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 1 (upper), including DARS2 gene (lower).
CUT and RUN Image 3: Helios (D4Z6D) Rabbit mAb Expand Image
CUT&RUN was performed with Jurkat cells and either Helios (D4Z6D) 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 ZNF687 promoter primers 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.