Anti-Cdk2 antibody [E304] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk2 antibody [E304] - BSA and Azide free (AB208697)

This data was developed using ab32147, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling Cdk2 with ab32147 at a concentration of 1/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab32147 anti-Cdk2 antibody [E304] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cdk2 antibody [E304] - BSA and Azide free (AB208697)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32147).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk2 antibody [E304] - BSA and Azide free (AB208697)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32147).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk2 antibody [E304] - BSA and Azide free (AB208697)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32147).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cdk2 antibody [E304] - BSA and Azide free (AB208697)

Intracellular 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32147).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cdk2 antibody [E304] - BSA and Azide free (AB208697)

Clone E304 (ab208697) has been successfully conjugated by Abcam. This image was generated using Anti-Cdk2 antibody [E304] (Alexa Fluor® 647). Please refer to ab206038 for protocol details.

ab206038 staining Cdk2 in HeLa cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab206038 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IP

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Immunoprecipitation - Anti-Cdk2 antibody [E304] - BSA and Azide free (AB208697)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32147).

All lanes:

Immunoprecipitation - Anti-Cdk2 antibody [E304] (<a href='/en-us/products/primary-antibodies/cdk2-antibody-e304-ab32147'>ab32147</a>)

Predicted band size: 34 kDa

false

• WB

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Western blot - Anti-Cdk2 antibody [E304] - BSA and Azide free (AB208697)

This WB data was generated using the same anti-Cdk2 antibody clone, E304, in a different buffer formulation (cat# ab32147).

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 4°C. 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.

All lanes:

Western blot - Anti-Cdk2 antibody [E304] (<a href='/en-us/products/primary-antibodies/cdk2-antibody-e304-ab32147'>ab32147</a>)

Predicted band size: 34 kDa

false