We use the ProteomeLab® IgY-12 High Capacity Primate-Optimized Proteome Partitioning Kit from Beckman Coulter for the removal of highly abundant proteins from serum samples which will be used in differential protein expression studies. As serum is problematic due to the low abundance of interesting proteins in comparison to the high abundance of some classical serum proteins, an effective removal of these abundant proteins is needed for many studies. The ProteomeLab® IgY-12 High Capacity Proteome Partitioning Kits are specifically designed to remove twelve highly abundant proteins from human/primate biological fluids, such as serum and plasma, and there is an additional kit that removes seven high abundant proteins from rodent sera. Kits are available in both spin column and LC column formats, depending upon the desired capacity per cycle. The observed capacity of human or other primate serum or plasma varies from 20 µL to 250 µL, depending on the kit.
Using the spin columns, we depleted human sera and also horse sera (which is not recommended by Beckman Coulter) and tested the removal efficiency in comparison to another commercially available IgG and albumin removal kit, as well as the DAC method. Removal of abundant proteins was most efficient with the ProteomeLab® IgY-12 High Capacity columns. A further advantage is that the abundant proteins are in the bound fraction of the column so there is no interference with albumin or IgG degradation products when working with the low abundance protein fraction afterwards, as we observed with other techniques.
Use of the columns is quite simple, the serum sample is first diluted with dilution buffer (tris buffered saline, no urea or other chaotropic agents are used in the buffers), and after a centrifugation step, the column is loaded with the diluted sample. After mixing the sample with the beads directly in the column, the column incubates for 15 minutes on an end-to-end-rotator. After a further centrifugation step, the low abundant protein fraction is in the flow through. The abundant proteins are stripped from the column afterwards. The last step is a regeneration of the column, which can be re-used around 100 times. The manufacturer guarantees at least 90% removal of fibrinogen, á2-Macroglobulin, á1-Acid Glycoprotein and IgM, 95% removal of IgA, Haptoglobin, Apolipoprotein A-I and Apolipoprotein A-II, and 99% removal of Albumin, IgG and Transferrin on up to 100 cycles of the column. Further on, Orosomucoid is removed from the plasma fraction by this column.
Although the kit is expensive, the cost for each removal of highly abundant proteins is reasonable because of the many times that the system can be reused. We have not yet performed 100 cycles with our column, but we have used it about 50 times without recognizing a drop in removal quantity or quality. The column also works fine with the horse sera and is the best highly abundant protein removal system of the 3 methods we tested.
ProteomeLab® IgY-12 High Capacity Primate-Optimized Proteome Partitioning Kits from Beckman Coulter
Efficient, reliable and fast removal of highly abundant proteins with good reproducibility. In comparison to other removal protocols, no degradation products of albumin or IgG were observed.
The Bottom Line
Highly effective removal of twelve highly abundant proteins with one column. Simple and fast protocol, columns can be reused often without loss of quality. No interference in further procedures with degradation products from removed proteins.