Very High Yields & Concentrations
PhyNexus Innovation is Improving Diagnostics
PhyNexus offers the diagnostics industry one of the most innovative, effective platforms available for diagnostics applications. First, PhyTip Columns can accommodate virtually any type of chemistry, standard or custom, to accommodate virtually any diagnostic application. Next, PhyTip Columns work extremely well with very low sample volumes (resin beds as small as 5 µL), as the back-and-forth flow of DFC drives the reaction to equilibrium and delivers very high target yields and concentrations; diagnostic method miniaturization without sacrificing target sample volumes. Finally, PhyTip Columns are designed for bench top and HTP automation, which allows for HTP analysis, test scaling, sample load adjustments, and consistent, reproducible results.
Miniaturized, automated diagnostic applications using PhyTip Columns and DFC are already delivering breakthrough results. Diagnostics customers use PhyTip Column technology for protein complex purification from serum prior to LC-MS analysis; for immunoaffinity chromatography of solid tissue membrane proteins prior to LC-MS analysis; and derivatized columns to capture an active nucleoprotein complex which performs on-column reverse transcription of oligo DNA for analysis by polyacrylamide gel and phosphorimager.
Two Powerful, Precise Methods of Using PhyTip Columns for Diagnostics
PhyTip Columns offer researchers designing clinical diagnostic applications two different, very powerful methods for screening and diagnostic applications. The first method is Capture, Purify & Detect. The second method, unique to PhyTip Columns, is On-column Reaction.
Capture, Purify & Detect
This method is the classic process of directly capturing, purifying, eluting and analyzing the diagnostic target of interest. The capture and detect process can be straight-forward by detecting the diagnostic analyte directly, or rather more subtle by first tagging an analyte with His, GST or some other visualizing agent to make it detectable. At times, a diagnostic analyte within a protein complex may have to be manipulated to be adequately detected, and PhyTip Columns can do such. In fact, because PhyTip Columns work with standard automated systems, the entire process of tagging, capture, purification and elution can be brought together into one integrated robotic workflow. Once the diagnostic analyte is captured, detection can be done by spectroscopy, mass spectrometry, or another appropriate method.
On-column Reactor
PhyNexus is unique in the ability to deliver an On-column Reactor capability using standard liquid handling instrumentation and driving a mobile sample phase across of solid phase contained within the tip of the column. Not only is this a very powerful tool, the method benefits from the small sample size required, the high target concentrations delivered, and the ease of automation all afforded by PhyTip Columns. In this method, the PhyTip Column serves as an enzymatic or chemical reactor. An enzyme or reacting reagent is immobilized on the resin bed of the PhyTip Column. The diagnostic target reacts with the immobilized reagent to produce a detectable analyte as a byproduct of the reaction. In this case, the diagnostic target itself is not detected, but rather the existence, effectiveness, and volume of the diagnostic target can be measured by the byproduct analyte produced.
Automation, Accuracy and Precision for Diagnostics
The simple fact is, virtually no diagnostic screen or test is viable without HTP automation compatibility. But equally important, no diagnostic screen or test will succeed without very concentrated, very high quality results from very limited sample sizes to ensure accurate diagnosis. Finally, the screen or test must be reliable and repeatable, every time. PhyNexus PhyTip Columns deliver automation, accuracy and precision. PhyNexus technology is exceptionally efficient for protein-to-protein and protein complex capture. A wide range of diagnostic targets can be captured for detection and measurement. PhyTip Columns are able to reproducibly manipulate small liquid volumes, and flow rates, reactions volumes and reagents can be introduced into any method with fine control so that the reactions produced are rugged and reproducible. These benefits lead to higher concentrations of the diagnostic targets, which means higher screen or test sensitivity. All with the advantages of repeatability, reliability and throughput that come with automation.
Detecting Tagged Protein Complex Markers
The cholesterol-protein complex (or generically, “protein complex”) consists of several proteins and lipids. The complex is highly dynamic and one or more of the proteins is in a constant bound or unbound. A biological sample, such as blood, can be collected from patients where this complex is present. The sample is subject to an in vitro exchange reaction where the blood is mixed with a His-tagged, recombinant version of the dynamic protein described. The result is a biological sample where the protein complex of interest has exchanged a native protein component for a His-tagged version. PhyTip Columns packed with IMAC resin are used to pull out this specific protein complex from the blood sample where it would bind to the PhyTip Column and contaminants would be washed. The resulting purified complex would be eluted in a small volume resulting in a high concentration. The components of the complex are analyzed by LC-MS with high reliability and reproducibility because the PhyTip Column reduced noise and enhances signal.
Immunoaffinity Chromatography for Solid Tissue Proteins
Quantitative extraction of intact, integral membrane protein from solid tissue is superior to current methods of soluble fragment analysis because there is no loss of information. Antibodies directed against specific integral membrane proteins are covalently attached to PhyTip Columns packed with activated resin. The PhyTip Columns, now transformed into immunoaffinity PhyTip Columns are used to capture integral membrane proteins from complex biological samples such as tissue preparations. The integral membrane proteins are loaded onto PhyTip Columns, washed to remove impurities and elute into a small volume of buffer for high concentration. The integral membrane proteins are analyzed by mass spectrometry to measure mass heterogeneity and the ratios of different isoforms.
On-column Reactor for Measuring Transcription Activity
The biological importance of telomerase is highlighted by the 2009 Nobel Prize in Medicine. Aging, cancer and regeneration are biological functions associated with telomerase. However, the presence of telomerase alone is not sufficient to determine a diagnostic relevance. Instead it is the functional activity of the telomerase that is importance. PhyTip Columns packed with Protein G were used to immobilize an antibody specific for a protein component of the human Telomerase Reverse Transcriptase (hTERT) nucleoprotein complex. The derivatized PhyTip Columns capture hTERT from tissue and cell lysated followed by sufficient washing to remove inhibitors and artifacts. The immobilized hTERT complex is active and able to perform reverse transcription of oligo DNAs while immobilized on the PhyTip Column. The results of the on-column assay are short DNAs polymerized by the hTERT complex and the DNAs can be analyzed by polyacrylamide gel and phosphorimager.
PhyNexus Advantages
| Compatible HTP Systems |
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| Agilent |
| Beckman |
| Caliper (Perkin Elmer) |
| Dynamic Devices |
| Hamilton |
| Perkin Elmer |
| Tecan |