Anti-Cdk2 抗体 [E304]

• WB

Lab

Western blot - Anti-Cdk2 antibody [E304] (AB32147)

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.

All lanes:

Western blot - Anti-Cdk2 antibody [E304] (ab32147)

Predicted band size: 34 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk2 antibody [E304] (AB32147)

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)

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cdk2 antibody [E304] (AB32147)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cdk2 antibody [E304] (AB32147)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk2 antibody [E304] (AB32147)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk2 antibody [E304] (AB32147)

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.

• IP

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Immunoprecipitation - Anti-Cdk2 antibody [E304] (AB32147)

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.

All lanes:

Immunoprecipitation - Anti-Cdk2 antibody [E304] (ab32147)

Predicted band size: 34 kDa

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• WB

Lab

Western blot - Anti-Cdk2 antibody [E304] (AB32147)

Western blot : Anti-CDK2 antibody [E304] (ab32147) staining at 1/2000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab32147 was shown to bind specifically to CDK2. A band was observed at 34 kDa in wild-type MCF7 cell lysates with no signal observed at this size in CDK2 knockout cell line ab282628. To generate this image, wild-type and CDK2 knockout MCF7 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Cdk2 antibody [E304] (ab32147)

Lane 1:

Wild-type MCF7 cell lysate at 10 µg

Lane 2:

CDK2 knockout MCF7 cell lysate at 10 µg

Lane 2:

Western blot - Human CDK2 knockout (hetero) MCF7 cell line (ab282628)

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

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• WB

Supplier Data

Western blot - Anti-Cdk2 antibody [E304] (AB32147)

All lanes:

Western blot - Anti-Cdk2 antibody [E304] (ab32147) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 20 µg

Lane 2:

Hela cell lysate at 20 µg

Lane 3:

K562 cell lysate at 20 µg

Lane 4:

293 cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 34 kDa

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• WB

Lab

Western blot - Anti-Cdk2 antibody [E304] (AB32147)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of HeLa whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab32147 (1/5000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab32147 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-Cdk2 antibody [E304] (ab32147) at 1/5000 dilution

All lanes:

HeLa whole cell lysate at 15 µg with NDFM/TBST

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

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Exposure time: 10s

• WB

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Western blot - Anti-Cdk2 antibody [E304] (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] (ab32147)

Predicted band size: 34 kDa

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• WB

Lab

Western blot - Anti-Cdk2 antibody [E304] (AB32147)

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.

All lanes:

Western blot - Anti-Cdk2 antibody [E304] (ab32147) at 1/1000 dilution

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:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 34 kDa

Observed band size: 34 kDa

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Exposure time: 4min

• WB

Supplier Data

Western blot - Anti-Cdk2 antibody [E304] (AB32147)

All lanes:

Western blot - Anti-Cdk2 antibody [E304] (ab32147) at 1/1000 dilution

All lanes:

NIH/3T3 cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 34 kDa

false

• WB

Supplier Data

Western blot - Anti-Cdk2 antibody [E304] (AB32147)

All lanes:

Western blot - Anti-Cdk2 antibody [E304] (ab32147) at 1/5000 dilution

Lane 1:

C6 cell lysate at 10 µg

Lane 2:

PC-12 cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 34 kDa

false

• WB

AbReview45219****

Western blot - Anti-Cdk2 antibody [E304] (AB32147)

All lanes:

Western blot - Anti-Cdk2 antibody [E304] (ab32147) at 1/1000 dilution

Lane 1:

Human osteosarcoma whole cell lysate - control, non-targeting siRNA at 20 µg

Lane 2:

Human osteosarcoma whole cell lysate - siRNA for CDK2 at 20 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG polyclonal at 1/2000 dilution

Predicted band size: 34 kDa

Observed band size: 34 kDa

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Exposure time: 2s

This image is courtesy of an Abreview submitted by Sonia Rocha