One particularly stubborn obstacle in proteomic research is the detection of very rare proteins. And, since the molar ratio of the most abundant protein to the rarest protein in a cell can approach 10
12, it is no small problem. One method to address this dilemma is to prefractionate the protein sample, such as a cell extract, into less complex mixtures. Each fraction is then subjected to one or more of several available techniques (e.g. 2-D gel electrophoresis, mass spectrometry) designed to display the expressed proteins. Alternatively, the fractions can be further fractionated into even simpler protein mixtures prior to analysis. Whatever the method of fractionation, it is certain that some sort of chromatography will be involved. Along these lines, Harvard Bioscience carries a line of exceptionally useful spin columns with a wide variety of packing materials.
The Amika spin columns come in 3 sizes: ultra-micro, micro, and macro for maximum sample volumes of 25, 75, or 150 µl, respectively. There is also a 96-well spin column with a 100-µl maximum sample volume for high throughput applications. The list of packing materials available is quite extensive and includes several flavors each of gel filtration, hydrophobic, hydrophilic, ion exchange, and specialty packings (such as activated charcoal and detergent removal media). Empty columns are also available.
All of the Amika spin columns are intended for single use. Since the price ranges from about $1.10 to $5.40 per column (or $105.00 to $540.00 for a 96-well plate), depending on the packing material, using these devices can be economical or fairly expensive. Fortunately, the convenience and time saving serve to offset much of the expense.
We have used the macro spin columns packed with C-4 media (Vydac) with good results to simultaneously concentrate dilute protein solutions and reduce urea contamination. The entire procedure, from equilibrating the columns to elution of the concentrated proteins takes less than an hour.
Overall, this is a well-designed and very useful product. Fractionation, and sub-fractionation of small volumes of complex protein mixtures prior to proteomic analyses can be accomplished simply, efficiently, and in a high throughput fashion. The one downside is the potential for things to get expensive as the sample number increases.
Michael Campa, Ph.D.
Asst. Research Professor of Radiology
Duke University Medical Center