Description
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.
Methanol Procedure for a 96-well plate, with nuclear counterstain: 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 by adding 100 µl of -20°C 90% methanol or -20°C BD™ Phosflow Perm Buffer III (Cat. No. 558050) to each well and incubating for 5 minutes at RT. 4. Remove the permeabilizer, and wash the wells twice with 100 μl of 1× PBS. 5. 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 in blocking buffer or Stain Buffer (FBS), and stain the cells by adding 50 µl of the diluted antibody to each well and incubating for 1 hour at RT. 7. Remove the diluted antibody, and wash the wells three times with 100 μl of 1× PBS. 8. Remove the PBS, dilute the second-step reagent in blocking buffer or Stain Buffer (FBS), and stain the cells by adding 50 µl of the diluted second-step reagent to each well and incubating for 1 hour at RT. 9. Remove the diluted second-step reagent, and wash the wells three times with 100 μl of 1× PBS. 10. 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. 11. View and analyze the cells on an appropriate imaging instrument. Bioimaging: For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp Western blot: For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml
Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
3. 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.
4. 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.
