Albumin and IgG make up about 60% of the total protein content of serum. This becomes very important in analysis techniques, such as 2D-gel electrophoresis, where the total amount of protein that can be analyzed in one experiment is limited. For example, a 200 µg serum protein load on a 2D gel is actually like loading 120 µg of albumin and IgG, and 80 µg of all the other proteins. Given that there is a concentration range of proteins in serum approaching 12 orders of magnitude, loading as much protein on a gel as possible is crucial if there is to be any hope of detecting even moderately scarce proteins. One way to increase the amount of potentially interesting (i.e. non-albumin and -IgG) proteins that can be analyzed on a 2D-gel is by removing the most abundant ones. Then, a 200 µg protein load is actually 200 µg, not 80 µg. So, by simply removing two abundant proteins, the capacity of the 2D gel has been increased 2.5 fold. In addition, the huge albumin and IgG spots are much reduced in size, allowing the visualization of proteins formerly obscured.
While it is certainly possible to home-brew a method to remove albumin and IgG from serum, chances are it would take a lot of optimization, characterization, and time before it could be used routinely in the lab. A much easier approach is to use the Albumin and IgG Removal Kit from GE Healthcare (formerly Amersham Biosciences). The kit consists of enough immunoaffinity resin and spin tubes to process 10 serum samples. With the recommended serum load of 15 µl, the company claims that >95% of albumin and >90% of IgG will be removed. It is possible to use larger volumes of serum, up to 45 µl, but smaller proportions of albumin and IgG will be removed.
One of the best features of the kit is its extraordinary simplicity. Serum and immunoaffinity resin are mixed and allowed to interact for at least 30 min at room temperature. Bound and free proteins are then separated with the spin filter. Toss the filter, keep the flow through, and you’re done, almost. What started as serum at around 60-70 mg protein/ml is now a relatively dilute solution containing around 0.5 mg protein/ml. For many applications, desalting and concentration may be required. I performed an acetone precipitation to desalt and concentrate using the protocol in the kit instructions and had very good recovery of protein.
The figure to the left shows a Coomassie stained 12% SDS-PAGE gel of 10 µg total serum protein (S) or 10 µg protein from albumin- and IgG-depleted serum (DS). The removal of albumin is essentially complete. It’s a little harder to see IgG removal as there are other immunoglobulins (IgM and IgA) that are not removed. The point is that many protein bands appear darker in the depleted sample than in the non-depleted sample. By extrapolation, we assume this effective concentration increase is also occurring with low abundance proteins that are not visible on this gel.
Although I haven’t tried it, if desired, it might be possible to elute the bound proteins from the immunoaffinity matrix and reuse it. With gentle conditions (i.e. pH manipulation), the antibodies on the column shouldn’t be harmed.
Overall, I have been very happy with this product. The only drawback is the expense.
Associate Research Professor
Department of Radiology
Duke University Medical Center