All lanes : Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : CCND2 knockout HEK293T cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 33 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?

Lanes 1-2: Merged signal (red and green). Green - ab207604 observed at 36 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab207604 Anti-Cyclin D2 antibody [EPR19659] was shown to specifically react with Cyclin D2 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267318 (knockout cell lysate ab257875) was used.  Wild-type and Cyclin D2 knockout samples were subjected to SDS-PAGE.  ab207604 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

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.

Flow Cytometry analysis of U-2 OS (Human bone osteosarcoma epithelial cell) cells labeling Cyclin D2 with purified ab207604 at 1/450 dilution (1.00 µg/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).

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

Lane 1 : Full length human Cyclin D2 recombinant protein
Lane 2 : Full length human Cyclin D1 recombinant protein

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 33 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

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

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : Caco-2 (Human colorectal adenocarcinoma cell line) whole cell lysate
Lane 3 : U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 33 kDa
Observed band size: 33 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

 

All lanes : Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

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


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

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.

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.