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

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CDK4 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 34 kDa
Observed band size: 34 kDa

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 ab255378 (knockout 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 (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 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) (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  (ab7291) at 1/1000 dilution and  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 ab150120 at 1/500 dilution.
-ve control 2: ab7291 at 1/1000 dilution followed by ab150077  at 1/500 dilution.

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

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Cdk4 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 34 kDa

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 (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.

Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/2000 dilution + NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L Peroxidase conjugated at 1/1000 dilution

Developed using the ECL technique.

Predicted band size: 34 kDa


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

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) (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 (ab7291) at 1/1000 dilution and 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 ab150120 at 1/500 dilution.
-ve control 2: ab7291 at 1/1000 dilution followed by ab150077  at 1/500 dilution.

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

Lane 1 : Mouse brain whole cell lysate
Lane 2 : Mouse heart whole cell lysate

Lysates/proteins at 10 µg per lane.

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: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/20000 dilution + C2C12 (Mouse myoblast cell line) whole cell lysate at 10 µg

Secondary
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: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

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

Lane 1 : C6 (Rat glial tumor cell line)
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line)

Lysates/proteins at 10 µg per lane.

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: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

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

Lane 1 : WT HAP1 cell lysate
Lane 2 : CDK4 knockout HAP1 cell lysate

Lysates/proteins at 20 µg per lane.

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

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 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.

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 (ab172730) instead of ab199728 in NIH/3T3 whole cell lysate.

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