Anti-Cyclin A antibody [E23.1] (ab38)

Western blot - Cyclin A antibody [E23.1] (ab38)

Predicted band size : 60 kDa


Western Blot of Swiss 3T3 cells showing presence of cyclin A in rapidly dividing cells (1) but absent in quiesent cells (2). Recombinant cyclin A (3). Molecular weight markers 114, 98, 68, 57, 43 and 32 kD.

Julian Gannon, CRUK.

Immunocytochemistry/ Immunofluorescence - Cyclin A antibody [E23.1] (ab38)

ICC/IF image of ab38 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab38, 5µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Immunoprecipitation - Cyclin A antibody [E23.1] (ab38)

Immunoprecipitation using ab38 at 4µg/mg whole cell lysate diluted in RIPA buffer and incubated with Protein G matrix at 4°C for 12 hours.

This antibody can immunoprecipitate Cyclin A from both Human (293T) and Rat (PC12) cell extract. The species assigned as Cyclin A runs at  ~55 kDa.

Lanes 1-3 = 293T

Lanes 4-6 = PC12

Lane 1: Cyclin A antibody IP

Lane 2: Control antibody IP

Lane 3: 293T whole cell lysate at 10%

Lane 4: Cyclin A antibody IP

Lane 5: Control antibody IP

Lane 6: PC12 whole cell lysate at 10%

The Western Blot antibody was ab38 at 2µg/ml

This image is courtesy of an anonymous Abreview

Western blot - Cyclin A antibody [E23.1] (ab38)

All lanes : Anti-Cyclin A antibody [E23.1] (ab38) at 2 µg/ml

Lane 1 : U2OS (Hu) whole cell lysate
Lane 2 : 293T (Hu) whole cell lysate
Lane 3 : Cos-7 (AGMk) whole cell lysate
Lane 4 : CHO (Hm) whole cell lysate
Lane 5 : PC12 (Rat) whole cell lysate
Lane 6 : 3T3 (Ms) whole cell lysate

Lysates/proteins at 30 µg per lane.

Secondary
HRP conjugated goat anti-mouse at 1/5000 dilution

Performed under reducing conditions.

Predicted band size : 60 kDa
Observed band size : 58 kDa (why is the actual band size different from the predicted?)
Additional bands at : 64 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 6 minutes

The membrane was blocked with 5% milk for 1 hour at 21°C prior to incubation with the primary antibody for 12 hours at 4°C.

This image is courtesy of an anonymous Abreview