GATA3 Antibody

• Catalog Number: orb654426
• Category: Antibodies
• Description: GATA3 Antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: FC, ICC, IF, IHC, WB
• Reactivity: Human
• Isotype: Rabbit IgG
• Immunogen: A synthetic peptide corresponding to a sequence of human GATA3 (HSYMDAAQYPLPEEVDVLFNID).
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Dilution range: Western blot, 0.25-0.5μg/ml, Human Immunohistochemistry (Paraffin-embedded Section), 0.5-1μg/ml, Human Immunocytochemistry/Immunofluorescence, 4μg/ml, Human Flow Cytometry, 1-3μg/1x106 cells, Human
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 50 kDa
• UniProt ID: P23771
• Storage: Store at -20˚C for one year from date of receipt. After reconstitution, at 4˚C for one month. It can also be aliquotted and stored frozen at -20˚C for six months. Avoid repeated freeze-thaw cycles.
• Note: For research use only
• Application notes: Tested Species: In-house tested species with positive results. Other applications have not been tested. Optimal dilutions should be determined by end users. Add 0.2ml of distilled water will yield a concentration of 500ug/ml.
• Expiration Date: 12 months from date of receipt.

Western blot analysis of GATA3 using anti-GATA3 antibody (orb654426). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel)/90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions. Lane 1: human Jurkat whole cell lysates. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-GATA3 antigen affinity purified polyclonal antibody (Catalog # orb654426) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # orb90503) with Tanon 5200 system. A specific band was detected for GATA3 at approximately 50KD. The expected band size for GATA3 is at 50KD.

IHC analysis of GATA3 using anti-GATA3 antibody (orb654426). GATA3 was detected in paraffin-embedded section of human mammary cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-GATA3 Antibody (orb654426) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

IF analysis of GATA3 using anti-GATA3 antibody (orb654426). GATA3 was detected in immunocytochemical section of HeLa cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (orb90553) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 4μg/mL rabbit anti-GATA3 Antibody (orb654426) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of 293T cells using anti-GATA3 antibody (orb654426). Overlay histogram showing 293T cells stained with orb654426 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-GATA3 Antibody (orb654426, 1μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (5-10μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.