All lanes : Anti-Cyclin D3/CCND3 antibody [DCS2.2] (ab28283) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CCND3 knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : HEK-293 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) at 1/10000 dilution

Predicted band size: 35 kDa
Observed band size: 35 kDa

Lanes 1-4: Merged signal (red and green). Green - ab28283 observed at 35 kDa. Red - loading control ab52901.

 ab28283 Anti-Cyclin D3/CCND3 antibody [DCS2.2]  was shown to specifically react with Cyclin D3 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264931 (knockout cell lysate ab257876) was used. Wild-type and Cyclin D3 knockout samples were subjected to SDS-PAGE. ab28283 and Anti-beta Tubulin [EP1331Y] - Microtubule Marker (ab52901) were incubated at room temperature for 2.5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing HeLa cells stained with ab28283 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab28283, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

ab28283 (4µg/ml) staining Cyclin D3/CCND3 in human pancreas, using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Cyclin D3/CCND3 (KO) knockout HAP1 whole cell lysate (20 µg)
Lane 3: HEK293 whole cell lysate (20 µg)
Lane 4: Jurkat whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab28283 observed at 33 kDa. Red - loading control, ab176560, observed at 50 kDa.

ab28283 was shown to specifically recognize CCND3 in wild-type HAP1 cells along with additional cross reactive bands. No bands was observed when CCND3 knockout samples were uexamined. Wild-type and CCND3 knockout samples were subjected to SDS-PAGE. Ab28283 and ab176560 (Rabbit anti alpha Tubulin loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10,000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Cyclin D3/CCND3 antibody [DCS2.2] (ab28283) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 3 : K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 35 kDa
Observed band size: 35 kDa
Additional bands at: 75 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 8 minutes