M-5 Muscarinic Receptor IgG Fraction Antibody – Rabbit Polyclonal

Immunocytochemistry: The purified IgG has been found to stain PLP fixed cultured cells known to express the muscarinic M5 receptor at a concentration of 100 ug/ml.

Western Blot: Western immunoblots using whole rat brain homogenate and cultured cells known to express the M5 receptor have been successful using this pooled antisera at a concentration of 50 ug/ml.

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 4% normal goat serum using TBS/Tween-20 buffer as diluent.
2. Wash x 2 with TBS/Tween-20.
3. Apply the IgG fraction after diluting to at least 50 ug/ml (Note: higher dilutions may be needed). Use 2% 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 minutes per wash cycle in TBS/Tween-20.
7. Develop color using the enhanced DAB reaction.