Anti-Cyclin D2 antibody [EPR19659]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D2 antibody [EPR19659] (AB207604)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-2 OS (Human bone osteosarcoma epithelial cell line) cells labeling Cyclin D2 with ab207604 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and weak cytoplasmic staining on U-2 OS cells. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab207604 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Cyclin D2 antibody [EPR19659] (AB207604)

Intracellular Flow Cytometry analysis of U-2 OS (Human bone osteosarcoma epithelial cell) cells labeling Cyclin D2 with purified ab207604 at 1/450 dilution (1.00μg/mL) (Red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) (black). Unlabeled control - Unlabelled cells (blue).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D2 antibody [EPR19659] (AB207604)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Caco-2 (Human colorectal adenocarcinoma cell line) cells labeling Cyclin D2 with ab207604 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and weak cytoplasmic staining on Caco-2 cells. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab207604 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-Cyclin D2 antibody [EPR19659] (AB207604)

Cyclin D2 was immunoprecipitated from 0.35 mg of U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate with ab207604 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab207604 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : U-2 OS whole cell lysate, 10μg (Input).

Lane 2 : ab207604 IP in U-2 OS whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab207604 in U-2 OS whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-Cyclin D2 antibody [EPR19659] (ab207604)

Predicted band size: 30 kDa,33 kDa

false

• WB

Lab

Western blot - Anti-Cyclin D2 antibody [EPR19659] (AB207604)

False colour image of Western blot : Anti-Cyclin D2 antibody [EPR19659] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab207604 was shown to bind specifically to Cyclin D2. A band was observed at 33 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in Ccnd2 knockout cell line ab267318 (knockout cell lysate ab257875). To generate this image, wild-type and Ccnd2 knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human CCND2 (Cyclin D2) knockout HEK-293T cell lysate (<a href='/en-us/products/cell-lysates/human-ccnd2-cyclin-d2-knockout-hek-293t-cell-lysate-ab257875'>ab257875</a>) at 20 µg

Lane 2:

Western blot - Human CCND2 (Cyclin D2) knockout HEK-293T cell lysate (<a href='/en-us/products/cell-lysates/human-ccnd2-cyclin-d2-knockout-hek-293t-cell-lysate-ab257875'>ab257875</a>)

Lane 2:

Western blot - Human CCND2 (Cyclin D2) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-ccnd2-cyclin-d2-knockout-hek-293t-cell-line-ab267318'>ab267318</a>)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

U-2 OS cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

false

• WB

Supplier Data

Western blot - Anti-Cyclin D2 antibody [EPR19659] (AB207604)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution

Lane 1:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 2:

Caco-2 (Human colorectal adenocarcinoma cell line) whole cell lysate at 10 µg

Lane 3:

U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-Cyclin D2 antibody [EPR19659] (AB207604)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

ab181602 was used as a GAPDH loading control.

We recommend using a higher sensitive ECL substrate to increase the band intensity.

All lanes:

Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution

Lane 1:

293T (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

U-2 OS (Human bone osteosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Human heart tissue lysate at 20 µg

Lane 4:

Human thyroid tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 33 kDa

Observed band size: 31 kDa

true

Exposure time: 40s

• WB

Lab

Western blot - Anti-Cyclin D2 antibody [EPR19659] (AB207604)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : CCND2 (Cyclin D2) knockout HAP1 whole cell lysate (20 μg)
Lane 3 : Hek293 whole cell lysate (20 μg)
Lane 4 : HeLa whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab207604 observed at 34 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab207604 was shown to recognize CCND2 (Cyclin D2) in wild type cells as signal was lost at the expected MW in CCND2 (Cyclin D2) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CCND2 (Cyclin D2) knockout samples were subjected to SDS-PAGE. ab207604 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)

Predicted band size: 33 kDa

false

• WB

Supplier Data

Western blot - Anti-Cyclin D2 antibody [EPR19659] (AB207604)

Blocking/Dilution buffer : 5% NFDM/TBST.

Full length human Cyclin D2 recombinant protein contains aa1-289 with a His-Tag. Full length human Cyclin D1 recombinant protein contains aa1-295 with a His-Tag. These two recombinant proteins were made in-house.

All lanes:

Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution

Lane 1:

Full length human Cyclin D2 recombinant protein at 0.01 µg

Lane 2:

Full length human Cyclin D1 recombinant protein at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 33 kDa

Observed band size: 35 kDa

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-Cyclin D2 antibody [EPR19659] (AB207604)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

false

Exposure time: 3min