Anti-Abi-1 抗体 (ab65828)

Western blot analysis of ABI1 using ab65828
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 µg of sample under reducing conditions.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with ab65828 at 0.5 µg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1/5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for ABI1 at approximately 60-65 kDa. The expected band size for ABI1 is at 55 kDa.

Immunofluorescence analysis of section of U2OS cells staining ABI1 with ab65828 at 5 µg/mL concentration. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. Blocking was done with 10% goat serum and incubated with the primary antibody overnight at 4°C. Secondary antibody Cy3 Conjugated Goat Anti-Rabbit IgG was used at 1/500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI.

Immunofluorescence analysis of paraffin-embedded section of human intestine cancer tissue staining ABI1 with ab65828 at 5 µg/mL concentration.
Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). Blocking was done with 10% goat serum and the tissue section was then incubated with the primary antibody overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI.

Immunofluorescence analysis of paraffin-embedded section of human breast cancer tissue staining ABI1 with ab65828 at 5 µg/mL concentration.
Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). Blocking was done with 10% goat serum and the tissue section was then incubated with the primary antibody overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI.

Flow cytometry analysis staining 4% paraformaldehyde-fixed permeabilized MCF-7 cells with ab65828 (Blue) at 1 µg/1x10⁶ cells concentration. Blocking was done using 10% normal goat serum, and the cells were incubated with the primary antibody for 30 minutes at 20°C. The secondary used was DyLight®488 conjugated goat anti-rabbit IgG at 5-10 µg/1x10⁶ cells, and the isotype control was rabbit IgG (Green) at 1 µg/1x10⁶ for 30 minutes at 20°C. Control used was unlabelled sample (Red).