Anti-Aurora A antibody [35C1]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Aurora A antibody [35C1] (AB13824)

This data was developed using the same antibody clone in a different buffer formulation without PBS and sodium azide (ab264552)

ab264552 staining Aurora A in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-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 ab264552 at 5µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• ICC/IF

AbReview7290****

Immunocytochemistry/ Immunofluorescence - Anti-Aurora A antibody [35C1] (AB13824)

ab13824, at 1/2000 dilution, detecting Aurora A (green) in Hela Cells in conjunction with a Goat anti-mouse secondary antibody conjugated to Cy3^®. Cells were fixed with methanol and counterstained with DAPI. Please refer to abreview for further details.

This image is courtesy of an Abreview submitted by Dr Kirk McManus

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Aurora A antibody [35C1] (AB13824)

Overlay histogram showing HeLa cells stained with ab13824 (red line). The cells were fixed with 80% methanol (5 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. The cells were then incubated with the antibody (ab13824, 2μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2μg/1x10⁶ cells ) used under the same conditions. Acquisition of >5,000 events was performed.

• WB

Lab

Western blot - Anti-Aurora A antibody [35C1] (AB13824)

This blot was produced using 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200v for 50 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab13824 over night at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution.

All lanes:

Western blot - Anti-Aurora A antibody [35C1] (ab13824) at 5 µg/mL

Lane 1:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 20 µg

Lane 2:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 45 kDa

Observed band size: 125 kDa,50 kDa,55 kDa

true

Exposure time: 20min