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Product Features
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12 Proteins comprise up to 96% of the protein mass in plasma
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Proteome Partitioning
Protein depletion has been used for some years to remove most of the albumin and/or IgG from biofluids such as plasma and serum prior to analysis, but it is clear that this alone is insufficient to enable progress to be made in biomarker discovery. The presence of highly abundant proteins significantly complicates the discovery process by masking the presence and limiting the detection of low abundance species. ProteomeLab IgY partitioning addresses this issue by reversibly capturing 12 of the more abundant proteins from human biofluids such as plasma and serum, yielding an enriched pool of low abundance proteins for further study. The captured proteins can also be easily recovered for investigation if required—hence the term partitioning rather than depletion.
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IgY base immunoaffinity chemistries will selectively partition the highly abundant proteins such as highlighted in the above illustration. The partitioned fractions can be taken to the next stage of the discovery process, such as multi-dimensional fractionation using the ProteomeLab PF 2D Protein Fractionation System or profiling using 2D PAGE (Polyacrylamide Gel Electrophoresis).
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This differential display map was generated by the ProteomeLab PF 2D software, allowing you to visualize the differences that exist between whole plasma versus the enriched/partitioned plasma. The red bands illustrate proteins that predominate in whole plasma—which in this case are effectively the 12 major proteins being partitioned out. The green bands represent proteins that predominate in the partitioned sample representing enrichment of the low to medium abundance proteins. All of these proteins are collected as liquid phase fractions ready for characterization after partitioning with an IgY-based immunoaffinity solution and fractionation by the PF 2D System.
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Advantages of IgY Chemistries
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IgY antibodies are produced by injecting an avian species (chicken) with highly purified mammalian protein antigens. IgY chemistries offer broader antigen-binding host range and cleaner capture than IgG capture methods because of the evolutionary distance between chicken and mammals.
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Broader antigen-binding host range
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Multiple Protein Alignment using Clustal X and TreeView. Numbers represent % amino acid similarity to HSA.
Due to the phylogenetic difference, mammalian proteins are often more immunogenic in birds than in mammals,* hence the broader antigen-binding host range. ProteomeLab IgY Chemistry effectively leverages this well-documented evolutionary advantage.
*Gassman, M., Thoemmes, P., Weiser, T., and Huebscher, U. (1990) Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J. 4, 2528-2532
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IgY (left) does not bind in a non-specific manner like IgG (right)
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Cleaner capture
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As the polyclonal IgYs are avian, the Fc region of the antibody does not bind mammalian complement factors, Rheumatoid Factor, IgM, Fc receptor and Protein A or G, a known issue related to capture with mammalian antibodies. Coupled with high specificity across multiple epitopes, you get very specific binding across species while achieving very clean capture with little background. The SDS-PAGE imaged below highlights whole serum run in lane #1, serum with just albumin partitioned out by ProteomeLab IgY-HSA SC chemistry in lane #2, and the isolated/partitioned albumin captured cleanly in lane #3. The partitioning efficiency of the reagents from human plasma is highlighted as well. To further improve capture, both primate-optimized 12-plex (IgY-12) and rodent-optimized 7-plex (IgY-R7) affinity solutions are available. |
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| Table 1: Partitioning Efficiency as Measured by ELISA |
| Target Protein |
Guaranteed % Removal |
Typical QC Results |
| Albumin |
> 99% |
99.6% |
| IgG |
> 99% |
99.1% |
| Transferrin |
> 99% |
99.1% |
| Fibrinogen |
> 90% |
96.7% |
| IgA |
> 95% |
99.0% |
| α2-Macroglobulin |
> 90% |
94.2% |
| IgM |
> 90% |
99.0% |
| α1-Antitrypsin |
> 95% |
99.7% |
| Haptoglobin |
> 95% |
99.3% |
| α1-Acid Glycoprotein |
> 90% |
99.1% |
| Apolipoprotein A-I |
> 95% |
99.2% |
| Apolipoprotein A-II |
> 95% |
99.2%
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Proteome Partitioning
Rather than employing a depletion strategy, proteome partitioning focuses on both the affinity capture of highly abundant proteins as well as the enrichment of the flow-through material.
Whether you are fractionating or profiling biofluid proteomes, the presence of highly abundant proteins will mask the identity of many of the medium to lower abundance species.
Now you can greatly enhance the detectability and identification of less abundant proteins through the selective partitioning of many of the highly abundant species. This process serves to both enrich the medium to low abundance proteins, while removing the deleterious masking effects generated by the overlapping peptide mass fingerprints from highly abundant proteins. In parallel, the captured highly abundant proteins are collected to allow for further analysis, as these proteins may also include a wealth of information. Either way, all protein fractions are preserved to allow you to monitor all aspects of the proteome.
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Imagine taking the equivalent of 50 mg of plasma or serum protein for a single fractionation experiment – this in fact is what you will be doing by combining the capacity of the ProteomeLab IgY-12 LC10 column with the fractionation power of the ProteomeLab PF 2D system. 1-3 cycles from the IgY-12 affinity column will enrich up to 5 mg of protein from 50 mg of plasma or serum protein – which the PF 2D system will fractionate into several hundred fractions using pI in the first dimension and hydrophobicity in the second dimension – bringing a whole new layer of proteins into focus. |
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ProteomeLab IgY High Capacity Solution
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Combining the innovative IgY chemistry with Beckman Coulter's proven separation technologies provides a convenient solution to simplify and enrich a plasma, serum or CSF proteome. Our high capacity solution includes the ProteomeLab PPS Proteome Partitioning System, the ProteomeLab SP Sample Preparation system and high capacity IgY Chemistry, capable of processing 250 µL of human and 200 µL of rat plasma/serum per cycle.
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The ProteomeLab PPS system consists of several components that integrate with the ProteomeLab PF 2D, providing an advanced pump, automated buffer switching and large volume fraction collection to implement the immunoaffinity-based proteome partitioning.
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ProteomeLab IgY Spin Column Solution
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Combining the innovative IgY chemistry with Beckman Coulter's proven separation technologies provides a convenient solution to simplify and enrich a plasma, serum or CSF proteome.
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The ProteomeLab IgY spin column solution includes the ProteomeLab SP Sample Preparation system, validated protocols in the form of standard operating procedures and the IgY Spin Column chemistry, configured as either IgY-12, which partitions 12 highly abundant proteins from human/primate biofluids or IgY-R7 which is optimized to partition seven highly abundant proteins from rodent biofluids.
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The IgY-12 High Capacity spin column solution is designed to process 20 µL human/primate serum or plasma per spin column cycle. The expected yield of a sample partitioned of the highly abundant proteins is about 160 µg. The IgY-R7 SC spin column has a capacity of 10 µL mouse or 15 µL rat serum/plasma per spin column cycle. The expected yield of a sample partitioned of the 7 highly abundant proteins is about 150 - 250 µg.
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ProteomeLab IgY Chemistries
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The ProteomeLab IgY-12 proteome partitioning chemistries are specifically designed to remove twelve highly abundant proteins from human/primate biological fluids such as serum, plasma, and cerebral spinal fluid (CSF). This technology enables removal of albinum, total IgG, α1-antitrypsin, IgA, IgM, transferrin, haptoglobin, α1-acid glycoprotein (orosmucoid), α2-macroglobulin, HDL (apolipoproteins A-I & A-II) and fibrinogen in a single step.
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The ProteomeLab IgY-R7 proteome partitioning chemistries are specifically designed to remove seven highly abundant proteins from rodent biological fluids such as serum, plasma, and cerebral spinal fluid (CSF). This technology enables removal of albumin, IgG, α1-antitrypsin, IgM, transferrin, haptoglobin and fibrinogen, in a single step.
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The ProteomeLab IgY chemistries come as complete kits, including reagents and optimized methods, plus one of the chemistry formats depicted below.
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ProteomeLab IgY
Spin Column
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Chemistry Formats
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The IgY spin column format is intended for more analytical scale analyses and utilizes centrifugation as the force for affinity separation. The IgY SC spin column format is available in IgY-12 and IgY-R7, as well as individual partitioning solutions for HSA, BSA, RSA, total IgG, fibrinogen, transferrin, and HDL.
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The IgY LC2 column format is a moderate capacity chemistry utilizing IgY microbeads packed into a 2 ml column bed with liquid chromatography used as the force for affinity separation. The High Capacity IgY-12 LC2 column has the capacity for 50 µL human/primate serum or plasma per LC column cycle, while the IgY-R7 LC2 column kit has a capacity of 20 µL mouse or 40 µL serum/plasma per LC column cycle.
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The IgY LC10 column format utilizes IgY microbeads packed into a 10 ml column bed with liquid chromatography used as the force for affinity separation. The IgY-12 LC10 column kit has a capacity of 250 µL human/primate serum or plasma per LC column cycle. The IgY-R7 LC 10 column has a capacity of 100 µL mouse or 200 µL rat serum/plasma per LC column cycle. The IgY-12 High Capacity LC10 column has an observed capacity of 250 µL serum/plasma per cycle.
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