When using a protein for labeling, functional studies, creating
in situ probes, cell studies, or multiple other uses, it is often necessary to purify or tag the protein of interest. If using a lot of the protein, it is usually beneficial to transiently transfect or create a stable cell line that will secrete that protein of interest. It can be a lot cheaper in the long run than paying a lot of money for a little vial of the protein, especially when mass quantities are needed to do some intense functional or behavioral studies. In creating cell lines, I most often tag the desired protein with an IgG-Fc to aid in purification. I have found it to be the simplest to purify and identify with Western blotting and a reliable anti-IgG Fc antibody.
Affinity chromatographic column purification is the easiest and most common method of purifying proteins from media or supernatant. The column is usually preloaded with a binding protein, in this case, Protein A, which will bind to the proteins as they are run through the column. Protein A is a component of the cell wall and will bind specifically to the Fc region of immunoglobulin molecules, with the greatest specificity to IgG. Four high affinity binding sites, high specificity, heat stability, and resistance to denaturation make Protein A very stable and effective as a purification tool. Pierce has packaged Protein A into columns, immobilizing it in gel-packed columns that offer good gel stability and excellent binding. There are special ImmunoPure Buffers included in the kit to enhance binding and facilitate the washing out of the bound protein after all other contaminants are run through the column.
The basic protocol requires equilibrating the column with binding buffer (letting the buffer run through the column), applying the diluted sample to the column and allowing it to flow through, washing the column with excess binding buffer, and eluting with elution buffer. Elution should be collected as fractions to preserve those fractions with the highest concentration. Fractions can be concentrated further using molecular weight ultra spin columns. Confirmation of protein of interest should then be performed, usually with a Western blot.
I have used these columns for purifying proteins from cell supernatant for several years. I usually obtain very pure protein, although the concentration is often low (this could also be due to the amount of protein secreted by the cells). It is important to use a serum-free cell media such as Opti-Mem that doesn’t contain FBS, as FBS will interfere with binding and purification. The biggest problem I often find is how slow the columns tend to run, especially during reuse. They can be stored in the fridge, however, and sample washing and elution can be performed over several days. Fortunately, the columns won’t run dry. It is easy to regenerate and store the columns for future use (although I make sure I am purifying the same Fc-tagged protein to avoid contamination). Although the columns have always worked with my samples, there is a decreased specificity for the IgG of certain species. It is important to check the protocol to make sure the IgG used will bind to the columns with enough specificity. Overall, the columns are easy-to-use, highly specific and highly effective in purifying the IgG protein or antibody of interest.
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Elizabeth Hawkes
Research Associate
Department of Ophthalmology
University of California, San Francisco
United States
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