Nicotinic Alpha 5 Receptor IgG Fraction Antibody – Rabbit Polyclonal

Immunocytochemistry: The pooled antisera have been found to stain fixed cells known to express the nicotinic alpha 5 receptor using ABC techniques at a concentration of 40 ug/ml. Cells and tissues should be fixed using PLP fixative

Western Blot: Western immunoblots using tissue homogenates have been successful at a concentration of 20 ug/ml and yield a single band at 75 kDa

Western Blot Protocol: 1. After SDS-PAGE (on either 4-15% gradient gels or single percentage gels. such at 10% gels) and electrophoretic transfer to PVDF membrane. block the membrane overnight with 2% normal goat serum using TBS/Tween-20 buffer.
2. Wash x 2 with TBS/Tween-20.
3. Apply the IgG fraction after diluting to at least 20 ug per ml (Note: higher dilutions may be needed). Use 2% normal goat serum in 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 TBS/Tween-20. Incubate 1-2 hours. Note: greater sensitivity may be achieved using ABC techniques.
6. Wash for 5 minutes x 4 in TBS/Tween-20.
7. Develop color using the enhanced DAB reaction.