PhyNexus, Inc.
3670 Charter Park Drive
Suite A
San Jose, CA, 95136
Website: http://www.phynexus.com
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Micro-Volume Scale Capture, Purification And Enrichment Of Proteins And Antibodies
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Introduction
The introduction of recombinant proteins that possess amino acid-tagged sites has resulted in a mechanism for many researchers to specifically extract proteins of interest from a mass of unwanted macromolecules in order to study the protein's structure and function. However, the isolation of recombinant proteins and antibodies for analysis by high sensitivity detection techniques such as mass spectrometry or protein biochips involves a time-consuming macro-scale process. For many applications, these same isolation processes result in an end product that may not retain functional integrity.
PhyNexus, Inc. (San Jose, Calif.) has developed a system that facilitates the production of sufficient quantities of purified and fully functional protein at a micro-volume scale. Using this system, with an associated liquid handling platform, researchers can rapidly capture, purify and enrich protein samples that are ready for further functional studies. Disposable PhyTip columns contain an aliquot of affinity resin embedded between two mesh retaining covers. Several resins are available (e.g. Protein A, Protein G, glutathione and IMAC i.e.Ni-NTA). These resins have different capture affinities and elution potentials. In general, a sample mixture of up to 1 ml of starting material can be introduced to a PhyTip column and at the end of the process 20 μl or less of high purified and enriched protein can be obtained for further analysis.
Materials and methods
PhyTip columns were used to purify and enrich specific proteins. IgG
was used as a model system with immobilized Protein A as the specific
affinity resin and a PhyTipT ME 1000 Purification System was used as the
delivery system. In order to precisely capture, purify and enrich the
desired protein the system was integrated into a PhyNexus Instrument
Stand. This allowed for all 8 channels of the system to accurately
perform the three separate steps.
Procedure
A series of experiments were performed with 1000+ PhyTip columns, which allow for up to 1 ml of initial protein mixture to be purified. Each column consists of 10 μl resin, embedded between the two mesh retaining covers. In the first step, as the protein mixture is withdrawn into the PhyTip, the protein of interest passes through the affinity resin bed and is captured, while non-specific proteins are not captured and remain in solution.
When the same 1 ml volume of liquid is expelled from the PhyTip column, the unbound proteins pass by the affinity resin and again are not captured. The second step, a wash step, removes any non-specifically bound proteins away from the affinity resin. Using a wash solution of PBS, 1 ml was first introduced into the PhyTip column, and then expelled. This process was repeated twice in order to ensure maximum purity of the end product. A second wash step was performed with water only, so as to ensure optimum elution conditions. The final step involved elution of the specific protein from the affinity resin. In order to obtain high enrichment of the target protein, this step is often performed with volumes as low as 10 - 20 μl of elution buffer. The elution buffer is a solution that rapidly and quantitatively removes the specific, functional protein from the affinity resin and back into solution.
Results
Recovery, selectivity and capacity. In order to demonstrate the selectivity of Protein A resin in PhyTip columns, an IgG recovery experiment was performed but with 10 μg IgG in the presence or absence of 5 mg of BSA in PBS. Under these conditions 65-70% of the starting quantity of IgG was routinely recovered. Also, no contaminating BSA was present in the final purified fraction as shown in Figure 1, lanes 7, 8, 10 and 11. Results indicate that 1000+ μl PhyTip columns are capable of preparing greater than 100 μg of purified IgG that can be eluted into a final volume of 20 μl. Processing results in fully intact antibody preparations suitable for further functional analysis. Typically the total time required to capture, purify and enrich a sample — with samples numbering from 1 to 96 at a time — is no more than 15 minutes from start to finish.
Summary
PhyTip columns provide rapid and efficient purification and enrichment proteins of interest from a background of highly contaminating material. The technique can easily be incorporated into a number of applications. For example, label-free detection technologies are becoming increasingly commonplace (e.g., instruments from Biacore (Piscataway, NJ), Applied Biosystems (Foster City, Calif.) SRU Biosystems (Woburn, Mass.) and Proterion (Piscataway, NJ)), and are being tapped for their ability to perform high-throughput kinetic based screening of hybridoma or phage-based antibody libraries, to obtain functional kinetic and equilibrium information — such as kas (onrates), kds (off-rates), Kd, etc. — at the primary screening stage.
The columns are also excellent precursors to ELISA-based primary screening procedures. By reducing false-positives through IgG enrichment and removal of the background proteins that potentially lead to non-specific interactions, one can reduce unnecessary, and costly, downstream processing. In addition, the PhyTip columns serve as economical and simple IgG enrichment and purification tools when determining affinity constants for ELISA-based Scatchard analyses within secondary screening procedures, as well as for FACS-based or cell- based secondary assay formats.
PhyTip columns available in a variety of formats for different instrument platforms, a variety of tip sizes for variable volume requirements and with a number of different capture resins for total flexibility.
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