Anti-Cyclin D3/CCND3 Antibody Picoband™

Figure 1. Western blot analysis of Cyclin D3 using anti-Cyclin D3 antibody (PB9103).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours.
lane 1: recombinant human Cyclin D3 protein 0.5ng.
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-Cyclin D3 antigen affinity purified polyclonal antibody (Catalog # PB9103) 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 Cyclin D3 at approximately 25KD. The expected band size for Cyclin D3 is at 25KD.

Figure 2. Western blot analysis of Cyclin D4 using anti-Cyclin D4 antibody (PB9103).
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 testis tissue lysate,
lane 2: rat thymus tissue lysate,
lane 3: rat lung tissue lysate,
lane 4: rat ovary tissue lysate,
lane 5: JURKAT whole cell lysate,
lane 6: A549 whole cell lysate,
lane 7: MCF-7 whole cell lysate,
lane 8: HELA whole cell lysate.
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-Cyclin D4 antigen affinity purified polyclonal antibody (Catalog # PB9103) 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 Cyclin D4 at approximately 33KD. The expected band size for Cyclin D4 is at 33KD.

Figure 3. Flow Cytometry analysis of K562 cells using anti-Cyclin D3 antibody (PB9103).
Overlay histogram showing K562 cells stained with PB9103 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Cyclin D3 Antibody (PB9103,1μg/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 4. Flow Cytometry analysis of THP-1 cells using anti-Cyclin D3 antibody (PB9103).
Overlay histogram showing THP-1 cells stained with PB9103 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Cyclin D3 Antibody (PB9103,1μg/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.