ab210823 stained in Raw264.7 cells. Untreated and LPS treated (1ug/ml, 24 hours) cells were fixed with 100% methanol (5min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab210823 at 5µg/ml overnight at +4°C then detected with an Alexa Fluor^® 488 goat anti-mouse secondary antibody (ab150117) at a 1/1000 dilution (shown in green). ab206369 (Rabbit monoclonal [EPR16774] to beta Tubulin Alexa Fluor® 594) was used as a counterstaining at a 1/200 dilution for 1hour at room temperature (pseudo-colored red). DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

Lane 1 : Anti-iNOS antibody [EPR16635] - Chimeric (ab210823) at 5 µg
Lane 2 : Anti-iNOS antibody [EPR16635] (ab178945) at 5 µg

All lanes : RAW 264.7 whole cell lysate treated with 0.1 µg/mL LPS for 6 hours

Lysates/proteins at 10 µg per lane.

Secondary
Lane 1 : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution
Lane 2 : Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 131 kDa
Observed band size: 131 kDa


Exposure time: 8 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 3% milk before being incubated with ab210823 (lane 1) and ab178945 (lane 2) overnight at 4°C. Antibody binding was detected using an anti-mouse (lane 1) and anti-rabbit (lane 2) antibody conjugated to HRP, and visualised using ECL development solution ab133406.