Anti-iNOS antibody (ab15323)

All lanes : Anti-iNOS antibody (ab15323) at 1 µg/ml

Lane 1 : Raw264.7
Lane 2 : Raw264.7 + LPS (1 ug per mL; 18 hours)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : IR- goat anti-rabbit (green) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 131 kDa

This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system under denaturing, reducing conditions. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with rabbit polyclonal to iNOS (ab15323; 1 ug x mL-1 (based on antibody concentration of 0.2mg/ml - lot GR126616-1) and the loading control mouse anti-GAPDH antibody (ab8245; 1:10000) overnight at 4°C. Antibody binding was detected using infrared (IR) labelled goat anti-rabbit (green; 1:10000) and IR-goat anti-mouse (red; 1:10000) for 1 hour at room temperature before imaging.

All lanes : Anti-iNOS antibody (ab15323) at 5 µg/ml

Lane 1 : Raw 264.7 whole cell lysate + PMA
Lane 2 : Raw 264.7 whole cell lysate + PMA + LPS + Brefeldin A

Lysates/proteins at 20 µg per lane.

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 131 kDa
Additional bands at: 140 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 8 minutes

All lanes : Anti-iNOS antibody (ab15323) at 5 µg/ml

Lane 1 : Human recombinant iNOS protein
Lane 2 : Mouse recombinant iNOS protein

Lysates/proteins at 0.1 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 131 kDa


Exposure time: 1 minute

Membrane was blocked in 2% milk for 1 hour at RT. Primary anitbody was incubated at 4°C overnight.

IHC image of iNOS staining in a section of formalin-fixed paraffin-embedded normal mouse lung performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab15323, 1/100 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

IHC image of iNOS staining in a section of formalin-fixed paraffin-embedded normal rat lung performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab15323, 1/100 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.