Anti-Cdk4 antibody [EPR17525]

10 product images

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  Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)

  Lanes 1- 2: Merged signal (red and green). Green - ab199728 observed at 34 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.
  

  ab199728 was shown to react with Cdk4 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human CDK4 knockout HeLa cell line ab255378 (knockout cell lysate Human CDK4 knockout HeLa cell lysate ab263780) was used. Wild-type HeLa and CDK4 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab199728 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (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.

  All lanes: Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/2000 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: CDK4 knockout HeLa cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 34 kDa

  Observed band size: 34 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR17525] (ab199728)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Cdk4 with ab199728 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic and nuclear staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).
  

  Tubulin is detected with Anti-alpha Tubulin antibody- Loading Control (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 1/500 dilution (red).
  The negative controls are as follows:
  -ve control 1: ab199728 at 1/250 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 at 1/500 dilution.
  -ve control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 at 1/500 dilution.

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  Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)

  Lanes 1 - 3: Merged signal (red and green). Green - ab199728 observed at 34 kDa. Red - loading control, ab18058, observed at 130 kDa.
  

  ab199728 was shown to specifically recognize Cdk4 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when Cdk4 knockout samples were examined. Wild-type and Cdk4 knockout samples were subjected to SDS-PAGE. ab199728 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 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.

  All lanes: Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/1000 dilution

  Lane 1: Wild-type HAP1 whole cell lysate at 20 µg

  Lane 2: Cdk4 knockout HAP1 whole cell lysate at 20 µg

  Lane 3: Wild-type HeLa whole cell lysate at 20 µg

  Predicted band size: 34 kDa

• 

  Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)

  Blocking/Dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/2000 dilution

  All lanes: NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG H&L Peroxidase conjugated at 1/1000 dilution

  Developed using the ECL technique.

  Predicted band size: 34 kDa

  Exposure time: 30s

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR17525] (ab199728)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Cdk4 with ab199728 at 1/250 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 1/500 dilution (green). Confocal image showing cytoplasmic, nuclear and membrane staining on HeLa cell line. The nuclear counter stain is DAPI (blue).
  

  Tubulin is detected with Anti-alpha Tubulin antibody - Loading Control (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 1/500 dilution (red).
  The negative controls are as follows:
  -ve control 1: ab199728 at 1/250 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 at 1/500 dilution.
  -ve control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 at 1/500 dilution.

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  Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)

  Blocking/Dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/10000 dilution

  Lane 1: Mouse brain whole cell lysate at 10 µg

  Lane 2: Mouse heart whole cell lysate at 10 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG H&L Peroxidase conjugated at 1/1000 dilution

  Developed using the ECL technique.

  Predicted band size: 34 kDa

  Observed band size: 34 kDa

  Exposure time: 1min

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  Immunoprecipitation - Anti-Cdk4 antibody [EPR17525] (ab199728)

  Cdk4 was immunoprecipitated from 1mg NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate with ab199728 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab199728 at 1/1000 dilution. Anti-Rabbit IgG (HRP) specific to the non-reduced form of IgG was used as secondary antibody at 1/1500 dilution.
  

  Lane 1: NIH/3T3 whole cell lysate, 10 (Input).

  Lane 2: ab199728 IP in NIH/3T3 whole cell lysate.

  Lane 3: Rabbit IgG,monoclonal [EPR25A]- Isotype Control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab199728 in NIH/3T3 whole cell lysate.

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.
  Exposure time: 10 seconds

  All lanes: Immunoprecipitation - Anti-Cdk4 antibody [EPR17525] (ab199728)

  Predicted band size: 34 kDa

  Observed band size: 34 kDa

• 

  Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)

  Blocking/Dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/20000 dilution

  All lanes: C2C12 (Mouse myoblast cell line) whole cell lysate at 10 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG H&L, Peroxidase conjugated at 1/1000 dilution

  Developed using the ECL technique.

  Predicted band size: 34 kDa

  Observed band size: 34 kDa

  Exposure time: 30s

• 

  Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)

  Blocking/Dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/2000 dilution

  Lane 1: C6 (Rat glial tumor cell line) at 10 µg

  Lane 2: PC-12 (Rat adrenal gland pheochromocytoma cell line) at 10 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG H&L, Peroxidase conjugated at 1/1000 dilution

  Developed using the ECL technique.

  Predicted band size: 34 kDa

  Observed band size: 34 kDa

  Exposure time: 15s

• 

  Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)

  ab199728 was shown to specifically react with CDK4 when CDK4 knockout samples were used. Wild-type and CDK4 knockout samples were subjected to SDS-PAGE. ab199728 and Anti-beta Actin antibody [mAbcam 8226] - Loading Control ab8226 (loading control to beta actin) were both diluted at 1/1000 and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

  All lanes: Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/1000 dilution

  Lane 1: WT HAP1 cell lysate at 20 µg

  Lane 2: CDK4 knockout HAP1 cell lysate at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit IgG H&L (IRDye® 800CW) at 1/10000 dilution

  Predicted band size: 34 kDa

  Observed band size: 34 kDa