Figure 1. Western blot analysis of 14-3-3 zeta/delta using anti-14-3-3 zeta/delta antibody (A01141-1).
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 Hela whole cell lysates,
Lane 2: human placenta tissue lysates,
Lane 3: human HepG2 whole cell lysates,
Lane 4: human A549 whole cell lysates,
Lane 5: human PANC-1 whole cell lysates,
Lane 6: human SK-OV-3 whole cell lysates,
Lane 7: human 22RV1 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-14-3-3 zeta/delta antigen affinity purified polyclonal antibody (Catalog # A01141-1) 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 # EK1002) with Tanon 5200 system. A specific band was detected for 14-3-3 zeta/delta at approximately 28KD. The expected band size for 14-3-3 zeta/delta is at 28KD.
Figure 10. Flow Cytometry analysis of U20S cells using anti-YWHAZ antibody (A01141-1).
Overlay histogram showing U20S cells stained with A01141-1 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-YWHAZ Antibody (A01141-1,1μg/1x106 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x106 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x106) used under the same conditions. Unlabelled sample (Red line) was also used as a control.
Figure 2. Western blot analysis of 14-3-3 zeta/delta using anti-14-3-3 zeta/delta antibody (A01141-1).
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: rat brain tissue lysates,
Lane 2: rat spleen tissue lysates,
Lane 3: rat lung tissue lysates,
Lane 4: rat liver tissue lysates,
Lane 5: mouse brain tissue lysates,
Lane 6: mouse spleen tissue lysates,
Lane 7: mouse lung tissue lysates,
Lane 8: mouse liver tissue lysates,
Lane 9: mouse kidney tissue 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-14-3-3 zeta/delta antigen affinity purified polyclonal antibody (Catalog # A01141-1) 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 # EK1002) with Tanon 5200 system. A specific band was detected for 14-3-3 zeta/delta at approximately 28KD. The expected band size for 14-3-3 zeta/delta is at 28KD.
Figure 3. IHC analysis of 14-3-3 zeta/delta using anti-14-3-3 zeta/delta antibody (A01141-1).
14-3-3 zeta/delta was detected in paraffin-embedded section of rat small intestine tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-14-3-3 zeta/delta Antibody (A01141-1) 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 # SA1022) with DAB as the chromogen.
Figure 4. IHC analysis of 14-3-3 zeta/delta using anti-14-3-3 zeta/delta antibody (A01141-1).
14-3-3 zeta/delta was detected in paraffin-embedded section of human lung cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-14-3-3 zeta/delta Antibody (A01141-1) 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 # SA1022) with DAB as the chromogen.
Figure 5. IHC analysis of 14-3-3 zeta/delta using anti-14-3-3 zeta/delta antibody (A01141-1).
14-3-3 zeta/delta was detected in paraffin-embedded section of human mammary cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-14-3-3 zeta/delta Antibody (A01141-1) 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 # SA1022) with DAB as the chromogen.
Figure 6. IHC analysis of 14-3-3 zeta/delta using anti-14-3-3 zeta/delta antibody (A01141-1).
14-3-3 zeta/delta was detected in paraffin-embedded section of rat kidney tissue . Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-14-3-3 zeta/delta Antibody (A01141-1) 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 # SA1022) with DAB as the chromogen.
Figure 7. IF analysis of 14-3-3 zeta/delta using anti-14-3-3 zeta/delta antibody (A01141-1).
14-3-3 zeta/delta was detected in immunocytochemical section of A431 cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-14-3-3 zeta/delta Antibody ((A01141-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
Figure 8. IF analysis of 14-3-3 zeta/delta using anti-14-3-3 zeta/delta antibody (A01141-1).
14-3-3 zeta/delta was detected in immunocytochemical section of A549 cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-14-3-3 zeta/delta Antibody ((A01141-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
Figure 9. Flow Cytometry analysis of A431 cells using anti-YWHAZ antibody (A01141-1).
Overlay histogram showing A431 cells stained with A01141-1 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-YWHAZ Antibody (A01141-1,1μg/1x106 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x106 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x106) used under the same conditions. Unlabelled sample (Red line) was also used as a control.
Figure 11. IHC analysis of 14-3-3 zeta/delta using anti-14-3-3 zeta/delta antibody (A01141-1).
14-3-3 zeta/delta was detected in paraffin-embedded section of mouse small intestine tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-14-3-3 zeta/delta Antibody (A01141-1) 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 # SA1022) with DAB as the chromogen.
