New Western Blotting Method

A new western blotting technique called Double Blotting is designed to overcome the high nonspecific binding of secondary antibodies during immunoblotting.

The procedure, which uses the Immobilon-P and PSQ transfer membranes of Millipore (Billerica, MA, USA), involves blotting proteins onto a sheet of Millipore's Immobilon membrane. The membrane is probed with the primary antibody and then assembled with a second membrane sheet. Both membrane sheets undergo a second blotting under acidic conditions.

This second blot allows the primary antibody molecules to desorb from their corresponding antigen and transfer onto the second membrane, whereas the antigen and the interfering proteins remain bound to the first membrane. The second membrane can then be probed by secondary antibodies without risk of nonspecific binding.

The Double Blotting method was developed by Dr. Francoise Lasne of the National Anti-Doping Laboratory in Chatanay-Malabry (France). The new method is suitable for a wide range of research applications, and is currently used for the detection of erythropoietin (EPO) in concentrated urine. Compared to traditional blotting, Double Blotting minimizes strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins.

Millipore's Immobilon-P and PSQ are polyvinylidene fluoride (PVDF) membranes, with a uniformly controlled pore structure and a high protein-binding capacity. In contrast to nitrocellulose materials, these membranes demonstrate reliable performance under the acidic conditions needed for Double Blotting, states Millipore.




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