Do you have any suggestion on electroblotting method?
The transfer is performed by the method of Matsudaira (J.Biol.Chem.;262,10035,1987).
- CAPS working solution:
- Stock Solution 10X
- 22.13g of CAPS in D.I. water 800 mL
- pH to 11.0 with NaOH
- adjust volume to 1000 mL
- Working Solution 1X - 200 mL of Stock 10X CAPS added to 200 mL MeOH and 1600 mL D.I. water
- Cut PVDF membrane to the size of the gel
- Wet membrane with methanol for a few seconds.
- Soak membrane in blotting buffer for five minutes
- Soak gel for five minutes in blotting buffer
- Soak pads and filters in blotting buffer and assemble the transblott unit
- Electroblott at 50 volts constant voltage for 20 minutes to one hour (for proteins from 80-100,000 and larger delete the methanol)
- Remove the membrane and the gel from the sandwich rinse with D.I. water
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03/31/2009