ICC/IF image of ab156173 stained MCF7 cells. The cells were 100% methanol fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab156173, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) HeLa and HepG2 cells at 5µg/ml.

Anti-GW182 antibody (ab156173) at 1 µg/ml + NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 210 kDa
Observed band size: 260 kDa why is the actual band size different from the predicted?
Additional bands at: 31 kDa, 50 kDa, 60 kDa, 75 kDa, 98 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes


This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab156173 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.