Anti-Cyclin D2 antibody [EPR19659] - BSA and Azide free

7 product images

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  Western blot - Anti-Cyclin D2 antibody [EPR19659] - BSA and Azide free (ab240377)

  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 - Anti-Cyclin D2 antibody [EPR19659] ab207604 observed at 34 kDa. Red - loading control, ab18058, observed at 130 kDa.

  Anti-Cyclin D2 antibody [EPR19659] 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. Anti-Cyclin D2 antibody [EPR19659] 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 Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Cyclin D2 antibody [EPR19659] ab207604).

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

  Predicted band size: 33 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D2 antibody [EPR19659] - BSA and Azide free (ab240377)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Caco-2 (Human colorectal adenocarcinoma cell line) cells labeling Cyclin D2 with Anti-Cyclin D2 antibody [EPR19659] ab207604 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (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 Caco-2 cells. The nuclear counter stain is DAPI (blue).

  Tubulin is detected with Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution (red).

  The negative controls are as follows:
  -ve control 1: Anti-Cyclin D2 antibody [EPR19659] ab207604 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution.
  -ve control 2: Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Cyclin D2 antibody [EPR19659] ab207604).

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  Flow Cytometry (Intracellular) - Anti-Cyclin D2 antibody [EPR19659] - BSA and Azide free (ab240377)

  Intracellular Flow Cytometry analysis of U-2 OS (Human bone osteosarcoma epithelial cell) cells labeling Cyclin D2 with purified Anti-Cyclin D2 antibody [EPR19659] 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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (black). Unlabeled control - Unlabelled cells (blue). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Cyclin D2 antibody [EPR19659] ab207604).
  

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  Immunoprecipitation - Anti-Cyclin D2 antibody [EPR19659] - BSA and Azide free (ab240377)

  Cyclin D2 was immunoprecipitated from 0.35 mg of U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate with Anti-Cyclin D2 antibody [EPR19659] ab207604 at 1/30 dilution. Western blot was performed from the immunoprecipitate using Anti-Cyclin D2 antibody [EPR19659] ab207604 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (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: Anti-Cyclin D2 antibody [EPR19659] ab207604 IP in U-2 OS whole cell lysate.

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of Anti-Cyclin D2 antibody [EPR19659] ab207604 in U-2 OS whole cell lysate.

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

  Exposure time: 3 minutes.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Cyclin D2 antibody [EPR19659] ab207604).

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

  Predicted band size: 30 kDa, 33 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D2 antibody [EPR19659] - BSA and Azide free (ab240377)

  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 Anti-Cyclin D2 antibody [EPR19659] ab207604 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (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 (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution (red).

  The negative controls are as follows:
  -ve control 1: Anti-Cyclin D2 antibody [EPR19659] ab207604 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution.
  -ve control 2: Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary at 1/1000 dilution.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Cyclin D2 antibody [EPR19659] ab207604).

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  Western blot - Anti-Cyclin D2 antibody [EPR19659] - BSA and Azide free (ab240377)

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

  Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as a GAPDH loading control.

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

  This data was developed using Anti-Cyclin D2 antibody [EPR19659] ab207604, the same antibody clone in a different buffer formulation.

  All lanes: Western blot - Anti-Cyclin D2 antibody [EPR19659] (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

  Developed using the ECL technique.

  Predicted band size: 33 kDa

  Observed band size: 31 kDa

  Exposure time: 40s

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  Western blot - Anti-Cyclin D2 antibody [EPR19659] - BSA and Azide free (ab240377)

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-Cyclin D2 antibody [EPR19659] 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 Human CCND2 (Cyclin D2) knockout HEK-293T cell line ab267318 (knockout cell lysate Human CCND2 (Cyclin D2) knockout HEK-293T 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  All lanes: Western blot - Anti-Cyclin D2 antibody [EPR19659] (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 (Human CCND2 (Cyclin D2) knockout HEK-293T cell lysate ab257875) at 20 µg

  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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 4: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 33 kDa

  Observed band size: 33 kDa

  False colour image of Western blot: Anti-Cyclin D2 antibody [EPR19659] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-Cyclin D2 antibody [EPR19659] 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 Human CCND2 (Cyclin D2) knockout HEK-293T cell line ab267318 (knockout cell lysate Human CCND2 (Cyclin D2) knockout HEK-293T 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.