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Purified Mouse anti-GATA4
Purified Mouse anti-GATA4
Western blot analysis using anti-human Gata4 antibody. Cell lysates were prepared from mouse embryonal carcinoma F9 (ATCC CRL-1720) cells treated with dibutryl cyclic AMP and retinoic acid.  Western Blot was probed using the GATA4 monoclonal antibody at concentrations of 0.02, 0.01, and 0.005 µg/ml (lanes 1, 2, and 3, respectively). GATA4 is identified as a band of 50 kDa.
Purified Mouse anti-GATA4

Immunofluorescent staining of hESC H9 derived cardiomyocytes (left panel) and mouse E12 embryo section (right panels). Left: Cells of the H9 cell line (WiCell, Madison, WI) were differentiated into cardiac mesoderm and plated on Permanox™ slides. The cells were fixed, permeabilized with BD Perm/Wash™ (Cat. No. 554723) then stained with Purified Mouse anti-GATA4 (2µg/mL) according to the Recommended Assay Procedure. The second step reagent was Alexa Fluor® 555 goat anti-mouse Ig (Invitrogen) (pseudo colored green). Cell nuclei were counterstained with Hoechst 33342 (pseudo colored blue). The image was captured on a BD Pathway™ 435 High-Content Bioimager System using a 20X objective, and merged using BD AttoVision™ software.

Right: Following antigen retrieval with BD Pharmingen™ Retrievagen A (pH 6.0) (Cat. No. 550524), the formalin-fixed paraffin-embedded mouse E12 embryo section was stained with anti-GATA4  monoclonal antibody followed by Alexa Fluor® 555 goat anti-mouse Ig (Invitrogen) (pseudo-colored red), and Alexa Fluor® 488 Mouse anti-Actin (Cat. No. 558623, pseudo colored green) according to the Recommended Assay Procedure for tissue sections.  The image was captured on a BD Pathway™ 435 High-Content Bioimager System as a 6x6 montage with a 10x objective. A collapsed Z-stack is shown: arrows indicate the developing heart and extra embryonic endoderm.

Western blot analysis using anti-human Gata4 antibody. Cell lysates were prepared from mouse embryonal carcinoma F9 (ATCC CRL-1720) cells treated with dibutryl cyclic AMP and retinoic acid.  Western Blot was probed using the GATA4 monoclonal antibody at concentrations of 0.02, 0.01, and 0.005 µg/ml (lanes 1, 2, and 3, respectively). GATA4 is identified as a band of 50 kDa.

Immunofluorescent staining of hESC H9 derived cardiomyocytes (left panel) and mouse E12 embryo section (right panels). Left: Cells of the H9 cell line (WiCell, Madison, WI) were differentiated into cardiac mesoderm and plated on Permanox™ slides. The cells were fixed, permeabilized with BD Perm/Wash™ (Cat. No. 554723) then stained with Purified Mouse anti-GATA4 (2µg/mL) according to the Recommended Assay Procedure. The second step reagent was Alexa Fluor® 555 goat anti-mouse Ig (Invitrogen) (pseudo colored green). Cell nuclei were counterstained with Hoechst 33342 (pseudo colored blue). The image was captured on a BD Pathway™ 435 High-Content Bioimager System using a 20X objective, and merged using BD AttoVision™ software.

Right: Following antigen retrieval with BD Pharmingen™ Retrievagen A (pH 6.0) (Cat. No. 550524), the formalin-fixed paraffin-embedded mouse E12 embryo section was stained with anti-GATA4  monoclonal antibody followed by Alexa Fluor® 555 goat anti-mouse Ig (Invitrogen) (pseudo-colored red), and Alexa Fluor® 488 Mouse anti-Actin (Cat. No. 558623, pseudo colored green) according to the Recommended Assay Procedure for tissue sections.  The image was captured on a BD Pathway™ 435 High-Content Bioimager System as a 6x6 montage with a 10x objective. A collapsed Z-stack is shown: arrows indicate the developing heart and extra embryonic endoderm.

製品詳細
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BD Pharmingen™
MGC126629, GATA-4
Mouse (QC Testing), Human (Reported)
Mouse BALB/c IgG1, κ
Human GATA4 Peptide
Western blot (Routinely Tested), Bioimaging (Tested During Development)
42 - 50 kDa
0.5 mg/ml
AB_1645188
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


推奨アッセイ手順

For more information, please refer to: http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml

and Bioimaging: http://www.bdbiosciences.com/pharmingen/protocols/Bioimaging_Certified.shtml

Recommended Protocol for Bioimaging:

1.        Seed the cells in appropriate culture medium at an appropriate cell density in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), and

        culture overnight to 48 hours.

2.        Remove the culture medium from the wells, and wash (one to two times) with 100 μl of 1× PBS.

3.        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).

4.        Remove the fixative from the wells, and wash the wells (one to two times) with 100 μl of 1× PBS.

5.        Permeabilize the cells using either cold methanol (a), Triton™ X-100 (b), or Saponin (c):

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.

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

c.        Add 100 µl of 1× Perm/Wash buffer (Cat. No. 554723) to each well and incubate for 15 to 30 minutes at RT.  Continue to use 1× Perm/Wash buffer for all subsequent wash and dilutions steps.

6.        Remove the permeabilization buffer from the wells, and wash one to two times with 100 μl of appropriate buffer (either 1× PBS or 1× Perm/Wash buffer, see step 5.c.).

7.        Optional blocking step:  Remove the wash buffers, and block the cells by adding 100 µl of blocking buffer BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or 3% FBS in appropriate dilution buffer to each well and incubating for 15 to 30 minutes at RT.

8.        Dilute the antibody to its optimal working concentration in appropriate dilution buffer.  Titrate purified (unconjugated) antibodies and second-step reagents to determine the optimal concentration.  If using a Bioimaging Certified antibody conjugate, dilute it 1:10.

9.        Add 50 µl of diluted antibody per well and incubate for 60 minutes at RT.  Incubate in the dark if using fluorescently labeled antibodies.

10.        Remove the antibody, and wash the wells three times with 100 μl of wash buffer.  An optional detergent wash (100 μl of 0.05% Tween in 1× PBS) can be included prior to the regular wash steps.

11.   If the antibody being used is fluorescently labeled, then move to step 12.  Otherwise, if using a purified unlabeled antibody, repeat steps 8 to 10 with a fluorescently labeled second-step reagent to detect the purified antibody.

12.        After the final wash, 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.

13.   View and analyze the cells on an appropriate imaging instrument.  

Recommended Assay Procedure for Tissue Sections:

1.        Harvest target organs or tissues and flush with PBS.

2.        Place the tissues in cassettes and fix with 10% neutral buffered formalin (Fisher SF100-4) for 16 hrs.

3.        Remove the fixative and proceed with processing and embedding in paraffin using standard protocols.*  

4.        Cut paraffin-embedded tissue sections (5 µm) using a microtome, adhere sections onto colorfrost slides (Fisher 12-550-17), and allow them

        to air dry.*

5.        Deparaffinize and re-hydrate the sections.*

6.        Treat with BD Retrievagen A solution (Cat. No. 550524) by heating the slides in a pressure cooker at 121°C for 15 minutes at 17 psi.*

7.        Allow the slides to cool to room temperature in the Retrievagen A, rinse the slides with tap water, and store in PBS prior to antibody

        staining.

8.        Sections can be simultaneously stained with a cocktail of multiple antibodies at pre-optimized concentration, for 2 hours at room

        temperature.

9.        Wash the sections in 1× PBS.

10.     If required label cellular DNA with 2 µg/ml Hoechst 33342 for 30 minutes, and wash with 1× PBS.

11.        Place cover slips on the sections using Aqua-Mount (Lerner Labs 13800).

12.        View and analyze the samples on an appropriate imaging instrument, such as the BD Pathway™ 435 High-Content Bioimaging System.  

        * for more details please see http://www.bdbiosciences.com/pharmingen/protocols/Paraffin_Sections.shtml

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
  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. Triton is a trademark of the Dow Chemical Company.
560327 Rev. 1
抗体の詳細
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L97-56

The L97-56 monoclonal antibody reacts with GATA4 (GATA-binding protein 4), a member of the GATA family of zinc finger-containing transcription factors that bind to the GATA nucleotide sequence. This ~50-kDa (observed MW) nuclear protein is expressed in mesodermal and definitive endodermal tissues such as the gastrointestinal tract, gonads, and heart. Genetic studies suggest that GATA4 regulates embryonic cardiac development: in mice, disruption of the GATA4 gene leads to defects in heart tube formation, while mutations of GATA4 are associated with atrial septal defects in humans. In the adult heart, GATA4 regulates differentiated gene expression. The roles of GATA4 in endocrine and reproductive functions were recently reviewed.

560327 Rev. 1
フォーマットの詳細
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
560327 Rev.1
引用&参考文献
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Development References (2)

  1. Oka T, Xu J, Molkentin JD. Re-employment of developmental transcription factors in adult heart disease. Semin Cell Dev Biol. 2007; 18(1):117-131. (Biology). View Reference
  2. Viger RS, Guittot SM, Anttonen M, Wilson DB, Heikinheimo M. Role of the GATA family of transcription factors in endocrine development, function, and disease. Mol Endocrinol. 2008; 22(4):781-789. (Biology). View Reference
560327 Rev. 1

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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.