Protein electroblotting

The blotting of proteins separated by two-dimensional polyacrylamide gel electrophoresis onto polyvinylidene difluoride (PVDF) membranes [3 and 20] has enabled the identification and characterization of proteins from complex biological samples. Transfer of the proteins can be carried out using several methods such as vacuum, capillary or electric field. Electroblotting is by far the most wide-spread technique which utilizes either vertical buffer tanks or semi-dry blotting. Both techniques can use either the Towbin or 3-[cyclohexamino]-1-propanesulfonic acid (CAPS) transfer buffers, depending on the need for minimal glycine contamination in post-transfer protein characterization. These two buffer systems are described here:

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

1. After second-dimensional electrophoresis, soak the gels in deionized water for 3 min.
2. 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.
3. Carry out electroblotting either in:
   1. 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 15^o C. Assemble the blotting sandwich as described in chapter 5 of this book.
   2. 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 15^o C or as described by the manufacturer.

       

CAPS buffer system

1. After second-dimensional electrophoresis, soak the gels in deionized water for 3 min.
2. 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.
3. Carry out electroblotting in either:
   1. 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 15^o C. Assemble the bloting sandwich as described in chapter 5 of this book.
   2. 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 15^o C or as described by the manufacturer.