45Calcium binding on Western

Dominik Haudenschild, 6-28-93
Boston notebook 3 pg 131

 

From papers:
Maruyam et al, J.Biochem. 95, 511-519 (1984)
Kirsch et al, FEBS 10444, Volume 294, Number 1,2 149-152

 

• Run SDS-PAGE gel and transfer onto Immobilon-P with tris/glycine/methanol buffer if protein is functional after SDS-PAGE

• If protein doesn’t bind calcium after being denatured, it is also possible to apply native protein to membrane with slot-blot method.
- 1 to 10µg per slot gives good signal

• Wash away transfer buffer and block membrane for 2 hours with ~3 changes of:
10mM Imidazole-HCl pH 6.8
60mM KCl
5mM MgCl2

• Add 2 mCi 45Ca per liter of above buffer, allow calcium to bind for 10-15 minutes

• Wash with 50% ethanol in water for 5 minutes

• Air dry completely then expose 12 to 24 hours at -80°C with intensifying screen

• Stain with Coomassie Blue to get size and intensity of protein bands