Lanes 1, 3 and 5: Wild-type HAP1 cell lysate (20 µg)
Lanes 2, 4 and 6: CDK2 knockout HAP1 cell lysate (20 µg)
Lanes 1 and 2: Green signal from target – ab32147 observed at 34 kDa
Lanes 3 and 4: Red signal from loading control – ab8226 observed at 42 kDa
Lanes 5 and 6: Merged (red and green) signal

ab32147 was shown to specifically react with CDK2 when CDK2 knockout samples were used. Wild-type and CDK2 knockout samples were subjected to SDS-PAGE. ab32147 and ab8226 (loading control to beta actin) were both diluted 1/1000 and incubated overnight at 4ºC. 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 1/10 000 dilution for 1 h at room temperature before imaging.

ab32147 staining Cdk2 in the HeLa cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody (1/200). ab150078 (1/500) an Alexa Fluor^® 555-conjugated Goat anti-rabbit IgG was used as the secondary antibody. Nuclei were counterstained with DAPI.

ab32147 staining Cdk2 in human tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed and paraffin-embedded, antigen retrieval was by heat mediation in Tris/EDTA buffer pH9. Samples were incubated with primary antibody (1/50). An undiluted HRP-conjugated mouse anti-rabbit IgG was used as the secondary antibody. Tissue counterstained with Hematoxylin. PBS was used in the negative control rather than the Primary antibody.

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling Cdk2 with purified ab32147 at 1/80 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

ab32147 (purified) at 1/40 immunoprecipitating Cdk2 from HeLa cells(Lane 1). Lane 2 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Lanes 1: Wild-type HAP1 cell lysate (20 µg)
Lanes 2: CDK2 knockout HAP1 cell lysate (20 µg)

Lanes 1 - 2: Merged signal (red and green). Green - ab32147 observed at 34 kDa. Red - loading control, ab8226, observed at 42 kDa or ab18058, observed at 130 kDa.

This western blot image is a comparison between ab32147 and a competitor's top cited rabbit polyclonal antibody.

ab32147 staining Cdk2 in human squamous cell carcinoma of cervix tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed and paraffin-embedded, antigen retrieval was by heat mediation in Tris/EDTA buffer pH9. Samples were incubated with primary antibody (1/50). An undiluted HRP-conjugated mouse anti-rabbit IgG was used as the secondary antibody. Tissue counterstained with Hematoxylin. PBS was used in the negative control rather than the Primary antibody.

All lanes : Anti-Cdk2 antibody [E304] (ab32147) at 1/5000 dilution

Lane 1 : C6 cell lysate
Lane 2 : PC-12 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), HRP-conjugated at 1/1000 dilution

Predicted band size: 34 kDa

Anti-Cdk2 antibody [E304] (ab32147) at 1/1000 dilution + NIH/3T3 cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), HRP-conjugated at 1/1000 dilution

Predicted band size: 34 kDa

All lanes : Anti-Cdk2 antibody [E304] (ab32147) at 1/1000 dilution

Lane 1 : Jurkat cell lysate
Lane 2 : Hela cell lysate
Lane 3 : K562 cell lysate
Lane 4 : 293 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), HRP-conjugated at 1/1000 dilution

Predicted band size: 34 kDa

All lanes : Anti-Cdk2 antibody [E304] (ab32147) at 1/1000 dilution (unpurified)

Lane 1 : Human osteosarcoma whole cell lysate - control, non-targeting siRNA
Lane 2 : Human osteosarcoma whole cell lysate - siRNA for CDK2

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG polyclonal at 1/2000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

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


Exposure time: 2 seconds

See Abreview

All lanes : Anti-Cdk2 antibody [E304] (ab32147) at 1/1000 dilution (unpurified)

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252) at 10 µg
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252) at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

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


Exposure time: 4 minutes

This blot was produced using a 10% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab32147 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.