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[Top] [Prev] [Next] [Bottom]Protein electroblotting
Gloves must be worn and all filter papers should be washed three times for 3 min in water and three times in transfer buffer. These two steps are important in order to avoid any protein or amino acid contamination.
Towbin buffer system
- After second-dimensional electrophoresis, soak the gels in deionized water for 3 min.
- Equilibrate the gels in a solution containing Tris (13 mM), glycine (100 mM) and methanol (10% v/v) for 30 min. At the same time, wet PVDF membranes in methanol for 1 min and equilibrate them in a solution containing Tris (13 mM), glycine (100 mM) and methanol (10% v/v) also for 30 min.
- Carry out electroblotting either in:
- a transfer tank with a solution containing Tris (13 mM), glycine (100 mM) and methanol (10% v/v) at 90 V constant voltage for 3 hours at 15oC. Assemble the blotting sandwich as described in chapter 5 of this book.
- or a semi-dry apparatus with a solution containing Tris (13 mM), glycine (100 mM) and methanol (20% v/v anodic side; 5% v/v cathodic side) at 1 mA/cm2 constant current for 3 hours at 15oC or as described by the manufacturer.
CAPS buffer system
- After second-dimensional electrophoresis, soak the gels in deionized water for 3 min.
- Equilibrate the gels in a solution containing 10 mM CAPS pH 11 for 30 min. At the same time, wet PVDF membranes in methanol for 1 min and equilibrate them in a solution containing 10 mM CAPS pH 11 and methanol (10% v/v) also for 30 min.
- Carry out electroblotting in either:
- a transfer tank with a solution containing 10 mM CAPS pH 11 and methanol (10% v/v) at 90 V constant voltage for 3 hours at 15oC. Assemble the bloting sandwich as described in chapter 5 of this book.
- or a semi-dry apparatus with a solution containing 10 mM CAPS pH 11 and methanol (20% v/v anodic side; 5% v/v cathodic side) at 1 mA/cm2 constant current for 3 hours at 15oC or as described by the manufacturer.
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