Western blotting

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Why FLASHBlot Transfer Buffer?

Background:

Since Towbin first described an electrophoretic protein transfer buffer in 1979, transfer buffers have remained unchanged and little has been done to try to achieve better tranfer performance.  In fact,  the mechanism and variables that drive the process of protein transfer largely remain a mystery. Most researchers use either a wet-tank or semi-dry transfer method; the gold standard is wet-tank transfer using an electrophoresis apparatus. There are three fundamental problems with the traditionally used Towbin buffer, including: 

a.  Protein transfer is a non-equilibrium process. The binding efficiency of proteins to the membrane and the rate of protein migration from the gel change over time during the transfer process. Since the process is non-equilibrium, protein charge changes during the transfer, and binding to the membrane depends on the amino acid composition of the protein more at the end of the transfer process than at the beginning of it.   

b. Different sized proteins migrate at different rates from the gel onto the membrane, with smaller proteins moving through the gel at a faster rate than larger proteins. By the time large proteins have reached the membrane, smaller proteins have often traveled through the membrane and are in solution.

c.  Transfer speed is slow.  Generally most researchers transfer their blots over 1-3 hours using a wet transfer method and Towbin buffer.   To reduce transfer times to 10 minutes, companies like Invitrogen and Bio-Rad offer semi-dry transfer apparatuses that cost up to $4000 with associated consumables and cartridges that cost $10s of dollars per experiment; a very expensive alternative.

 

Our Solution:  FlashBlot Transfer Buffer   

 

Advansta has developed a new buffer formulation that addresses all three of the above problems.  Our buffer creates conditions where proteins of different sizes 

 

travel through the gel onto the membrane at the same rate, so large and small proteins are represented more uniformly on the membrane.  Furthermore, Advansta's buffer creates an environment where proteins have improved efficiency of binding to the membrane.  Finally, the buffer enables fast transfer onto the membrane in less than 15 to 20 minutes.

Check out Advansta´s new blot on western blogging..

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..and perhaps the Wiki might provide some useful tips as well!

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