Ultrafiltration: Fast and Efficient Elution of Proteins from Polyacrylamide Gels

Ultrafiltration is a technique based on membrane separation that has been applied to the separation of dissolved small molecules. When a force, such as centrifugal force, is applied to such molecules, those smaller than the pore size of the membrane will pass through it while larger molecules will be retained.

Nanosep™ centrifugal devices have been designed for application of this technique in manipulation of both proteins and DNA. They are equipped with specially prepared membranes having different molecular weight cut-offs (MWCO). The choice of the correct MWCO (see table 1) allows for efficient concentration of protein solutions and also for fast and effective salt removal, buffer exchange, and protein fractionation. These devices thus provide a fast and inexpensive alternative to commonly used methods for the elution and concentration of proteins that have been seperated on polyacrylamide gels and, due to its gentle nature, this elution method is especially effective for biologically-active molecules and preparations of protein complexes.

Table of the selection guide based on Molecular Weight Cutoff

"The amount of starting polyacrylamide gel material is the limiting factor for high protein recovery."

Reducing the amount of polyacrylamide not only increases the yield of eluted protein but also reduces the centrifugation time significantly, a factor of consideration when eluting biologically-active proteins. This method has been applied successfully to the elution of enzymatically-active protein mixtures in their native state, under both nondenaturing and denaturing conditions, with no significant loss of activity. The amount of sodium dodecyl sulfate (SDS) in the elution buffer can be empirically determined to increase recovery while preserving biological activity. Ultrafiltration is, therefore, a fast and gentle method that is ideal for the elution of biologically-active proteins (figure 1).

Figure of the protocol for the elution of proteins

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