Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR7875] (ab133463) at 1/1000 dilution (purified) + HeLa cell lysate treated with UV at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded human colon with purified ab133463 at a working dilution of 1 in 75. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Direct ELISA antibody dose-response curve using purified ab133463 at 0-1000 ng/ml. Antigen concentration of 1000 ng/mL. An alkaline phosphatase-conjugated goat anti-rabbit IgG (H+L) (1/2500) was used as the secondary antibody.

Direct ELISA antibody dose-response curve using purified ab133463 at 0-1000 ng/ml. Antigen concentration of 1000 ng/mL. An alkaline phosphatase-conjugated goat anti-rabbit IgG (H+L) (1/2500) was used as the secondary antibody.

Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR7875] (ab133463) at 1/2000 dilution (purified) + C6 cell lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR7875] (ab133463) at 1/2000 dilution (purified) + NIH/3T3 cell lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Dot blot analysis of CDK1+CDK2+CDK3+CDK5 (pY15) phospho peptide (lane 1) and CDK1+CDK2+CDK3+CDK5 non-phospho peptide (lane 2) labelling CDK1+CDK2+CDK3+CDK5 (phospho Y15) with unpurified ab133463 at a dilution of 1/1000. A peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/2500).

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 10 seconds.

Saso2 cells were incubated at 37°C for 24 hours with vehicle control (0 μM) and different concentrations of Amifostine (ab141060). Decreased expression of CDK1 (phospho Y15) (unpurified ab133463) in Saso2 cells correlates with an increase in Amifostine concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with unpurified ab133463 at 1 μg/ml and ab32384 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 and visualised using ECL development solution.

Immunohistochemical analysis of paraffin embedded human breast carcinoma tissue labelling CDK1+CDK2+CDK3+CDK5 with unpurified ab133463 at 1/50 dilution. Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR7875] (ab133463) at 1/1500 dilution (unpurified) + NIH/3T3 cell lysate at 1/1000 dilution

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR7875] (ab133463) at 1/1500 dilution (unpurified) + C6 cell lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Equilibrium disassociation constant (K_D)
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All lanes : Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR7875] (ab133463) at 1/1000 dilution (unpurified)

Lane 1 : HeLa cell lysate
Lane 2 : HeLa cell lysate treated with UV

Lysates/proteins at 10 µg per lane.

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

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

Immunohistochemical staining of paraffin embedded human colon with unpurified ab133463 at a working dilution of 1 in 50. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.