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Abundant Protein Removal with the Enchantâ„¢ Multi-Protein Affinity Separation Kit
Sponsored,vendor-submitted protocol   Sponsored by Pall Life Sciences    Published in December 2006 (p.25)

Over the past decade, the interest in proteomics research has increased dramatically. Meaningful proteomics data rely on the development of reproducible and rapid methods; this methodology has been as challenging as the biological studies. Sample preparation has received a great deal of focus with particular emphasis on plasma and serum, two of the most readily accessible biological fluids having direct bio-chemical relevance. The concentrations of individual proteins in these samples range from >10 mg/mL to <1 pg/mL. As a result, the most abundant proteins often mask other proteins present in relatively low concentrations. Removal of the most abundant proteins is one of the few options for scientists interested in detecting the moderate to low abundancy proteins using mass spectrometry (MS) or two dimensional gelbased (2D gel) proteomic analytical methods. The removal of abundant proteins must be balanced with the desire to minimize sample handling to limit protein loss while maintaining reproducibility and ease-of-use characteristics.

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Pall Life Sciences offers a new kit for the depletion of human serum albumin (HSA) and human IgG. The kit utilizes a protein-based specific capture method to achieve maximal depletion of target protein and minimal loss of other proteins. Depletion of HAS and IgG is typically >98%.

Depletions take approximately 20 minutes from start to finish. The Enchant Multi-Protein Affinity Separation Kit uses Nanosep® devices (loaded with HSA and/or IgG depleting resins), which are disposable, thereby eliminating the possibility of cross-contamination. Each column is designed for depletion of 50 µL of plasma or serum, resulting in more depleted samples per column than most other commercially available kits. The increased amount of protein allows for greater flexibility in the number and type of analysis performed after the depletion step. The resins are mixed by the user, therefore it is easy to alter the ratios of HSA and IgG depletion resin when working with unusual samples containing higher or lower than normal concentrations of these proteins (e.g., specific diseased states). Additionally, the use of specific ligands for the capture of HSA and IgG overcomes the limitations of the commonly used blue dyes (e.g., Cibacron* Blue) for albumin removal, which are known to capture many other proteins, and protein A for antibody capture, which shows widely varying degrees of affinity for the antibody isotypes and sub-types. This results in incomplete removal of some sub-types of IgG.

Pall Life Sciences offers a protocol for serial or combined depletion of these two abundant serum/plasma proteins under native or denaturing conditions. The latter being a novel application of these stable immunochemical ligands leading to reduced nonspecific binding of serum/plasma proteins to the retained target protein. This new ligand-based depletion method is highly specific, disposable, cost-effective, and flexible.

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