Anti-Cyclin D3/CCND3 antibody [DCS2.2]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D3/CCND3 antibody [DCS2.2] (AB28283)

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.

• Flow Cyt

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Flow Cytometry - Anti-Cyclin D3/CCND3 antibody [DCS2.2] (AB28283)

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 22°C. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. 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.

• WB

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Western blot - Anti-Cyclin D3/CCND3 antibody [DCS2.2] (AB28283)

All lanes:

Western blot - Anti-Cyclin D3/CCND3 antibody [DCS2.2] (ab28283) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 10 µg

Lane 2:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Lane 3:

K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 33 kDa

Observed band size: 35 kDa,75 kDa

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Exposure time: 8min

• WB

Lab

Western blot - Anti-Cyclin D3/CCND3 antibody [DCS2.2] (AB28283)

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 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 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.

All lanes:

Western blot - Anti-Cyclin D3/CCND3 antibody [DCS2.2] (ab28283) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CCND3 knockout HeLa cell lysate at 20 µg

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

HEK-293 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 33 kDa

Observed band size: 35 kDa

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• WB

Lab

Western blot - Anti-Cyclin D3/CCND3 antibody [DCS2.2] (AB28283)

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:

Western blot - Anti-Cyclin D3/CCND3 antibody [DCS2.2] (ab28283)

Predicted band size: 33 kDa

Observed band size: 33 kDa

false