Signal transducer and activator of transcription 3
Anwendungsbeschreibung:
Application Notes: Western blot, 0.1-0.5 µg/ml, Human, Mouse, Rat Immunohistochemistry (Paraffin-embedded Section), 2-5µg/ml, Human Immunocytochemistry/Immunofluorescence, 5 µg/ml, Human Flow Cytometry(Fixed), 1-3 µg/1x106 cells, Human, Rat. Add 0.2ml of distilled water will yield a concentration of 500ug/ml
Flow Cytometry analysis of PC-12 cells using anti-STAT3 antibody. Overlay histogram showing PC-12 cells (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-STAT3 Antibody (1 µg/1x10 6 cells) for 30 min at 20C. DyLight488 conjugated goat anti-rabbit IgG (5-10 µg/1x10 6 cells) was used as secondary antibody for 30 minutes at 20C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10 6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used.
IHC analysis of STAT3 using anti-STAT3
Flow Cytometry analysis of SiHa cells using anti-STAT3 antibody. Overlay histogram showing SiHa cells (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-STAT3 Antibody (1 µg/1x10 6 cells) for 30 min at 20C. DyLight488 conjugated goat anti-rabbit IgG (5-10 µg/1x10 6 cells) was used as secondary antibody for 30 minutes at 20C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10 6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used.
IF analysis of STAT3 using anti-STAT3 antibody and anti-Beta Tubulin antibody. STAT3 was detected in immunocytochemical section of A549 cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 µg/mL rabbit anti-STAT3 Antibody and mouse anti-Beta Tubulin antibody overnight at 4C. DyLight488 Conjugated Goat Anti-Rabbit IgG and DyLight594 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
IF analysis of STAT3 using anti-U2OS antibody and anti-Beta Tubulin antibody. STAT3 was detected in immunocytochemical section of U2OS cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 µg/mL rabbit anti-STAT3 Antibody and mouse anti-Beta Tubulin antibody overnight at 4C. DyLight488 Conjugated Goat Anti-Rabbit IgG and Cy3 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
Western blot analysis of STAT3 using anti-STAT3 antibody. 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 30 ug of sample under reducing conditions. Lane 1: human HEL whole cell lysates, Lane 2: human A549 whole cell lysates, Lane 3: human Hacat whole cell lysates, Lane 4: human U251 whole cell lysates, Lane 5: human K562 whole cell lysates, Lane 6: human SiHa whole cell lysates, Lane 7: human RT4 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA 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-STAT3 antigen affinity purified polyclonal antibody at 0.5 µg/mL overnight at 4C, 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 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. Specific bands were detected for STAT3 at approximately 88 kDa. The expected band size for STAT3 is at 88 kDa.
Western blot analysis of STAT3 using anti-STAT3 a
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