Revision 8

#5852Store 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, IHC-P, IF-IC, FC-FP, ChIP, C&R

REACTIVITY:

H M

SENSITIVITY:

Endogenous

MW (kDa):

48

Source/Isotype:

Rabbit IgG

UniProt ID:

#P23771

Entrez-Gene Id:

2625

Product Information

Product Usage Information

For optimal ChIP 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
Immunohistochemistry (Paraffin) 1:1600 - 1:6400
Immunofluorescence (Immunocytochemistry) 1:800 - 1:3200
Flow Cytometry (Fixed/Permeabilized) 1:200 - 1:800
Chromatin IP 1:100
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.

Specificity / Sensitivity

GATA-3 (D13C9) XP® Rabbit mAb recognizes endogenous levels of total GATA-3 protein.

Species Reactivity:

Human, Mouse

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 Tyr63 of human GATA-3 protein.

Background

GATA proteins comprise a group of transcription factors that are related by the presence of conserved zinc finger DNA-binding domains, which bind directly to the nucleotide sequence core element GATA (1-3). There are six vertebrate GATA proteins, designated GATA-1 to GATA-6 (3).

GATA-3 is a critical regulator of development of various systems in both mouse and human (4). GATA-3 mouse embryos die between E11 and E12 due to growth retardation and deformities in the brain and spinal cord (5). The function of GATA-3 has been extensively studied in T cell development and has recently been shown to be a downstream target of Notch in Notch-mediated differentiation of TH2 cells (6,7). It is expressed in both hematopoietic and non-hematopoietic tissues, including the kidney, skin, mammary gland, and central nervous system (8-10). Decreased expression of GATA-3 in luminal breast cancer is associated with poor clinical outcome. GATA-3 expression level may therefore be a promising prognostic biomarker (11). Haploinsufficiency of GATA-3 results in Barakat syndome in humans, a condition characterized by sensorineural deafness and renal dysplasia (12).

  1. Ko, L.J. and Engel, J.D. (1993) Mol Cell Biol 13, 4011-22.
  2. Merika, M. and Orkin, S.H. (1993) Mol Cell Biol 13, 3999-4010.
  3. Lowry, J.A. and Atchley, W.R. (2000) J Mol Evol 50, 103-15.
  4. Debacker, C. et al. (1999) Mech Dev 85, 183-7.
  5. Pandolfi, P.P. et al. (1995) Nat Genet 11, 40-4.
  6. Ho, I.C. et al. (2009) Nat Rev Immunol 9, 125-35.
  7. Amsen, D. et al. (2007) Immunity 27, 89-99.
  8. Grote, D. et al. (2008) PLoS Genet 4, e1000316.
  9. Kaufman, C.K. et al. (2003) Genes Dev 17, 2108-22.
  10. Kouros-Mehr, H. et al. (2006) Cell 127, 1041-55.
  11. Chou, J. et al. (2010) J Cell Physiol 222, 42-9.
  12. Van Esch, H. et al. (2000) Nature 406, 419-22.

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 IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) 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.
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.

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 8
#5852

GATA-3 (D13C9) XP® Rabbit mAb

Western Blotting Image 1: GATA-3 (D13C9) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from CEM and SH-SY5Y cells using GATA-3 (D13C9) XP® Rabbit mAb.
Immunohistochemistry Image 1: GATA-3 (D13C9) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using GATA-3 (D13C9) XP® Rabbit mAb (left) or Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Immunohistochemistry Image 2: GATA-3 (D13C9) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded cell pellets, MCF-7 (left) or HUVEC (right), using GATA-3 (D13C9) XP® Rabbit mAb.
Immunofluorescence Image 1: GATA-3 (D13C9) XP® Rabbit mAb Expand Image
Confocal Immunofluorescent analysis of MCF7 (left) and HUVE (right) cells using GATA-3 (D13C9) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).
Flow Cytometry Image 1: GATA-3 (D13C9) XP® Rabbit mAb Expand Image
Flow cytometric analysis of THP-1 cells (blue, negative) and MCF7 cells (green, positive) using GATA-3 (D13C9) 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.
Flow Cytometry Image 2: GATA-3 (D13C9) XP® Rabbit mAb Expand Image
Flow cytometric analysis of THP-1 cells (blue) and MCF7 cells (green) using GATA-3 (D13C9) XP® Rabbit mAb.
Chromatin Immunoprecipitation Image 1: GATA-3 (D13C9) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from NK-92 cells and either GATA-3 (D13C9) 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 human CCL4 promoter primers, SimpleChIP® Human IFN-γ Promoter Primers #13051, 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: GATA-3 (D13C9) XP® Rabbit mAb Expand Image
CUT&RUN was performed with NK-92 cells and either GATA-3 (D13C9) XP® Rabbit mAb or GATA-3 (E2N1Y) Mouse mAb #96098, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across SH3RF3, a known target gene of GATA-3 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: GATA-3 (D13C9) XP® Rabbit mAb Expand Image
CUT&RUN was performed with NK-92 cells and either GATA-3 (D13C9) XP® Rabbit mAb or GATA-3 (E2N1Y) Mouse mAb #96098, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 2 (upper), including SH3RF3 (lower), a known target gene of GATA-3 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: GATA-3 (D13C9) XP® Rabbit mAb Expand Image
CUT&RUN was performed with NK-92 cells and either GATA-3 (D13C9) 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 RBM15 promoter primers, human SH3RF3 intron 1 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.