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Alexa Fluor® 488 Mouse anti-GATA3
Alexa Fluor® 488 Mouse anti-GATA3
Immunofluorescent staining of human breast adenocarcinoma.  MCF-7 cells (ATCC HTB-22) were cultured, fixed, permeabilized with cold methanol, stained with Alexa Fluor® 488 Mouse anti-GATA3 monoclonal antibody (pseudo-colored red, which appears pink when co-localized with the blue), and counter-stained with Hoechst 33342 (pseudo-colored blue) according to the Recommended Assay Procedure.  The images were captured on a BD Pathway™ 435 Bioimager System with a 20x objective and merged using BD Attovision™ software. We have observed somewhat dimmer staining when using Triton X-100 for permeabilization (see Recommended Assay Procedure).
Immunofluorescent staining of human breast adenocarcinoma.  MCF-7 cells (ATCC HTB-22) were cultured, fixed, permeabilized with cold methanol, stained with Alexa Fluor® 488 Mouse anti-GATA3 monoclonal antibody (pseudo-colored red, which appears pink when co-localized with the blue), and counter-stained with Hoechst 33342 (pseudo-colored blue) according to the Recommended Assay Procedure.  The images were captured on a BD Pathway™ 435 Bioimager System with a 20x objective and merged using BD Attovision™ software. We have observed somewhat dimmer staining when using Triton X-100 for permeabilization (see Recommended Assay Procedure).
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BD Pharmingen™
Human, Mouse (QC Testing), Human, Mouse (Reactivity Confirmed in Development), Rat (Predicted)
Mouse BALB/c IgG1, κ
Conserved peptide between the trans-activation and DNA-binding domains of human, mouse and rat GATA3
Bioimaging (Routinely Tested)
5 µl
AB_1645303
Aqueous buffered solution containing BSA, protein stabilizer, and ≤0.09% sodium azide.
RUO


Preparation and Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

推奨アッセイ手順

1.        Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), and

        culture overnight.

2.        Remove the culture medium from the wells, and fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and incubating for 10 minutes at room temperature (RT).

3.        Remove the fixative from the wells, and permeabilize the cells using either cold methanol or Triton™ X-100:

a.        Add 100 µl of -20°C 90% methanol or -20°C BD™ Phosflow Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.

OR

b.        Add 100 µl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.

Triton is a trademark of The Dow Chemical Company.

4.        Remove the permeabilizer, and wash the wells twice with 100 μl of 1× PBS.

5.        Optional blocking step:  Remove the PBS, and block the cells by adding 100 µl of blocking buffer (3% FBS in 1× PBS) or BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well and incubating for 30 minutes at RT.

6.        Remove the blocking buffer, dilute the antibody conjugate 1:10 in blocking buffer or Stain Buffer (FBS), and stain the cells by adding 50 µl of the diluted antibody conjugate to each well and incubating for 1 hour at RT.

7.        Remove the diluted antibody conjugate, and wash the wells three times with 100 μl of 1× PBS.

8.        Remove the PBS, and counter-stain the nuclei by adding 100 μl of a 2 μg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

9.        View and analyze the cells on an appropriate imaging instrument.  Recommended filters for the BD Pathway™ instruments are:

Instrument                        Excitation                Emission                Dichroic

BD Pathway 855                488/10                515 LP                Fura/Fitc

BD Pathway 435                482/35                536/40                FF506

Product Notices

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. This reagent has been pre-diluted for use at the recommended Volume per Test when following the Recommended Assay Procedure. A Test is typically ~10,000 cells cultured in a well of a 96-well imaging plate.
  3. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  6. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
560077 Rev. 1
抗体の詳細
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L50-823

GATA3 (GATA binding protein 3) is a member of the GATA family of transcription factors. This ~50-kDa nuclear protein regulates the development and subsequent maintenance of multiple tissues. GATA3 is involved in the development of T lymphocytes (regulates T cell receptor subunit gene expression) and the differentiation of mature T cells to become Th2 cells. The expressed levels of normal or mutant GATA3 are also associated with the behaviors of various cancer cells including estrogen receptor-positive breast carcinoma cells.

The L50-823 monoclonal antibody recognizes human and mouse GATA3.

This antibody conjugate is routinely tested and optimized for Bioimaging.  For flow cytometry, we recommend these alternate products:                  

                                        Fluorochrome conjugate        Size                Catalog No.

                                        Alexa Fluor® 488                        50 tsts        560163

                                        Alexa Fluor® 647                        50 tests        560068

                                        PE                                                50 tests        560074

560077 Rev. 1
フォーマットの詳細
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Alexa Fluor™ 488
Alexa Fluor™ 488 Dye is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 517-nm. Alexa Fluor™ 488 is designed to be excited by the Blue laser (488 nm) and detected using an optical filter centered near 520-nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 488
Blue 488 nm
494 nm
517 nm
560077 Rev.1
引用&参考文献
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Development References (8)

  1. Asselin-Labat M-L, Sutherland KD, Barker H, et al. Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nat Cell Biol. 2006; 9:201-209. (Biology).
  2. Kouros-Mehr H, Slorach EM, Sternlicht MD, Werb Z. GATA-3 maintains the differentiation of the luminal cell fate in the mammary gland. Cell. 2006; 127:1041-1055. (Biology).
  3. Marine J, Winoto A. The human enhancer-binding protein Gata3 binds to several T-cell receptor regulatory elements. Proc Natl Acad Sci U S A. 1991; 88(16):7284-7288. (Biology).
  4. Steenbergen RDM, OudeEngberink VE, Kramer D, et al. Down-regulation of GATA-3 expression during human papillomavirus-mediated immortalization and cervical carcinogenesis. Am J Pathol. 2002; 160(6):1945-1951. (Biology). View Reference
  5. Usary J, Llaca V, Karaca G, et al. Mutation of GATA3 in human breast tumors. Oncogene. 2004; 23(46):7669-7678. (Biology). View Reference
  6. Yang Z, Gu L, Romeo P-H, et al. Human GATA-3 trans-activation, DNA-binding, and nuclear localization activities are organized into distinct structural domains. Mol Cell Biol. 1994; 14(3):2201-2212. (Biology). View Reference
  7. Zheng W, Flavell RA. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell. 1997; 89(4):587-596. (Biology). View Reference
  8. van Esch H, Groenen P, Nesbit MA, et al. GATA3 haplo-insufficiency causes human HDR syndrome. Nature. 2000; 106:419-422. (Biology). View Reference
すべて表示する (8) 表示項目を減らす
560077 Rev. 1

Please refer to Support Documents for Quality Certificates


Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.