All lanes : Anti-CDK1 antibody [A17] (ab18) at 5 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 4 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 5 : RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) Whole Cell Lysate
Lane 6 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Rabbit Anti-Mouse IgG H&L (HRP) (ab97046) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 34 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?


Exposure time: 4 minutes

This blot was produced using a 4-12% 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 2% Bovine Serum Albumin before being incubated with ab18 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution.

Ab18 staining human skin. Staining is localized to the cytoplasm and nucleus.
Left panel: with primary antibody at 4 ug/ml. Right panel: isotype control.
Sections were stained using an automated system (Dako PT Link), at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffer, EDTA pH 9.0. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Overlay histogram showing MCF7 cells stained with ab18 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab18, 0.5µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Immunohistochemical analysis of Human lung cancer tissue, staining CDK1 with ab18 at 1/100 dilution.