NOS Antibody – Monoclonal

Western Blot: Western immunoblots resulted in a single band being detected at ~ 130 kDa at a dilution of 1:1000

Immunofluorescent: This monoclonal antibody has been found to stain cells induced to produce iNOS at a 1:1600 dilution. The ability of this monoclonal antibody to bind to iNOS in fixed cells was examined using two different cell lines. A-172 (a human glioblastoma cell line) and RAW 264.7 (a mouse macrophage cell line). The cells were cultured for 2 days in normal medium and then induced to produce iNOS by treatment for 16 hours with cytokine/LPS mixture. Following the treatment. the cells were washed x 4 and fixed in either 70% or 100% acetone. They were reacted for 60 minutes with the ascites fluid. and then with FITC-conjugated goat anti-mouse IgG. The immunofluorescent staining pattern was observed using epifluorescense microscopy.

Immunofluorescent: This monoclonal antibody has been found to stain cells induced to produce iNOS at a 1:1600 dilution. The ability of this monoclonal antibody to bind to iNOS in fixed cells was examined using two different cell lines. A-172 (a human glioblastoma cell line) and RAW 264.7 (a mouse macrophage cell line). The cells were cultured for 2 days in normal medium and then induced to produce iNOS by treatment for 16 hours with cytokine/LPS mixture. Following the treatment. the cells were washed x 4 and fixed in either 70% or 100% acetone. They were reacted for 60 minutes with the ascites fluid. and then with FITC-conjugated goat anti-mouse IgG. The immunofluorescent staining pattern was observed using epifluorescense microscopy.

Western Blot Protocol: 1. After SDS-PAGE (on either 4-15% gradient gels or single percentage gels. such at 7.5% gels) and electrophoretic transfer to PVDF membrane. block the membrane overnight with 4% normal goat serum in 1:5 diluted evaporated goat milk. using TBS/Tween-20 buffer as diluent.
2. Wash x 2 with TBS/Tween-20.
3. Apply the mouse monoclonal antibody after preparing a 1:1000 dilution. Use 2% normal goat serum in 1:5 diluted evaporated goat milk as buffer for the primary antibody. Dilute the condensed goat milk with TBS/Tween-20. Let the primary antibody bind for 2-4 hours.
4. Wash x 3 with TBS/Tween-20.
5. Apply affinity purified HRP goat anti-rabbit IgG antiserum diluted 1:2500 (dilution may vary depending upon supplier) in 2% normal goat serum in 1:5 diluted evaporated goat milk (use TBS/Tween-20 to dilute the goat milk). Incubate 1-2 hours. Note: greater sensitivity may be achieved using ABC techniques.
6. Wash x 3 and then soak the membrane overnight in a fairly large volume of TBS/Tween-20.
7. Develop color using the DAB reaction or the enhanced DAB reaction.
Note: If goat milk is not available(usually it can be found in grocery stores). you can substitue 4% normal goat serum