Hinos [1137-1153] Antibody – Rabbit Polyclonal

Immunocytochemistry: This antiserum has been found to stain fixed cultured cells known to express hiNOS by indirect immunofluorescence at a dilution of 1:1000 to 1:5000

Western Blot: Western immunoblots using homogenate from cells expressing hiNOS resulted in a single band being detected at ~ 130 kDa at a dilution of 1:1000.

Western Blot Protocol: 1. After SDS-PAGE (on either 4-15% gradient gels or single percentage gels. such at 12% gels) and electrophoretic transfer to PVDF membrane. block the membrane overnight with 2% normal goat serum in TBS/Tween-20 buffer.
2. Wash x 2 with TBS/Tween-20.
3. Apply the rabbit polyclonal antibody after dilution to at least 1:1000 (Note: higher dilutions may be needed). Use 1% normal goat serum in TBS/Tween-20 as buffer for the primary antibody. 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 1% normal goat serum in TBS/Tween-20. Incubate 1-2 hours. Note: greater sensitivity may be achieved using ABC techniques.
6. Wash x 4 for 5 min per wash cycle with TBS/Tween-20.
7. Develop color using the enhanced DAB reaction.