Applications Proteomics & Plasmid 2017-03-21T06:01:37+00:00

With the ever-growing DNA sequence information, proteomics is one of the most important disciplines for characterizing gene function and for providing insight into the mechanisms of biological processes in a high-throughput mode.  It is now possible to examine the expression of more than 1000 proteins using mass spectrometry technology coupled with various separation methods.

PhyNexus Automated Protein Purification

Automated Protein Purification or Plasma DNA Workflow with PhyTip Chromatography Tip Technology 

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phynexus phytip blue resin 3

The mission of PhyNexus is to provide a high performance system for small scale protein separations to life science researchers worldwide. PhyNexus flexible technology enables optimized preparation of 1-96 samples at a time using both Dynamic Devices automated Liquid Handling Platforms, the Oasis and Lynx, that provide fully unattended and scaleable operation.

phytip bidirectional

PhyNexus has patented chromatography tip technology for high throughput affinity chromatography for proteins and plasmid purification.  PhyTip columns are the most efficient and cost effective way to purify recombinant proteins, antibodies, and plasmids.   Their unique design allows bidirectional flow (see arrow in figure) of the sample and buffers through the column to maximize the use of the resin by increasing capture and elution of the target protein, leading to the highest yield possible. The dead volume is minimized with low surface area, thickness and pore volume of frits.  The design of the screens surrounding the resin allows elution volumes as low as 10 µL, and therefore high concentration product.  The high efficiency bidirectional washes lead to the highest possible sample purity.  Affinity purification can be performed with any resin of choice.  Higher performance is obtained than with any other resin purification method.

The Dynamic Devices Lynx liquid handler is used with the 96 Channel 1000 µL Head to perform protein purifications using the PhyTip columns. The system can use either 200 µL, 300 µL, 1000 or 1250 µL tips on the same head within the same run for pipetting large or small volumes.

The drive system of the head makes it a perfect fit for the PhyTip application since the drive can aspirate and dispense at extremely slow speeds, from 2-5 µL/sec, giving the sample the capture time needed to expose it to the solid phase matrix in order for optimal binding and elution.

With a Lynx liquid handling footprints from 36 to 66 inches, the Lynx LM900 has 30 positions for processing PhyTip columns. Deck layouts include:

PhyTip 1000 µL Tip Station
PhyTip Drying Station
Standard 1000 µL Tips
Deepwell Sample Plates
Deepwell Elution Plates
Column Prep Reagent Reservoir
Deepwell Wash Buffer 1
Deepwell Wash Buffer 2
Elution Buffer Reservoir

PhyNexus PhyTip Automated Lynx Deck

Deepwell plates are used to prevent cross contamination between samples during washing protocols and a Setup Run method can transfer Wash Buffers 1 & 2 from a reservoir to the deepwells and then removed to make more room for to process the samples with the PhyTip columns.

For more walk away time, our Lynx LM1200 offers 42 positions to process PhyTip columns. Combine the extra deck positions with 1-2 liter ‘feeder’ reservoirs to hold the wash buffers and triple the capacity. Additional configurations include dual 96 1000 µL Heads to process PhyTip simultaneously and gripper tools to move plates on and off the deck from storage containers and microplate readers, and a virtual ‘manufacturing line’ can be realized.

phynexus phytip pink resin 1 px300phynexus phytip blue resin 2 px300phynexus phytip blue resin 1 px300PhyTip Lynx Dryig Station

 Superior concentration, purity and activity of recovered protein or plasmid:

PhyTip columns employ a Virtual Heart-Cut Elution™ process to produce the highest possible concentration and purity of product.  In conventional affinity chromatography, the center of the eluted peak (heart cut of the peak) is captured to produce the highest concentrated product at the highest possible purity.

The mission of PhyNexus is to provide a high performance system for small scale protein separations to life science researchers worldwide. PhyNexus flexible technology enables optimized preparation of 1-96 samples at a time using both Dynamic Devices automated Liquid Handling Platforms, the Oasis and Lynx, that provide fully unattended and scaleable operation.

phynexus hc_exphynexus mab

Dynamic Devices Liquid Handling Robot 1-96 parallel processing

PhyTip column technology is a unique walk away bench-top separation process controlled with a high level of precision for life science researchers who prepare protein samples or plasmid DNA for analysis. By utilizing industry standard resins, it allows for the unattended parallel processing of a range of samples and volumes providing the highest flexibility in meeting the needs of a diversity of applications. Columns can be used to process sample volumes from 10 µL to 25 mL (sample volumes exceeding the tip column volume are processed as a series of fractions during the capture step).

The leading and trailing edges of the chromatography peak are not collected to remove impurities. The center of the peak is collected through the heart cut process to achieve high concentrations at high purity. It is an effective technology but tedious and very expensive to operate in parallel.

Virtual Heart-Cut technology is used to achieve superior protein concentration and purity. First, the affinity functional groups in the resin of the pipette tip column are fully loaded to capacity. This is accomplished by using back-and-forth flow of sample so that even slow interacting proteins have adequate contact time to be taken up by the resin. As many sample aliquots as desired can be loaded onto the column. In the case of limited sample, small column beds can be used. A fully loaded resin bed has the potential to produce a Virtual Heart-Cut, high concentration peak if eluted properly (described later).

High purity is achieved in the next step. Thin, low dead volume and low surface area column frits allow complete washing of the resin bed with a very high wash volume to resin volume ratio (up to 50 times) to thoroughly remove nonspecific bound contaminants. The frits are hydrophilic and have as much as 100 times less surface area compared with conventional frits so there is less surface area for contaminants to bind. High wash volume ratios and low column surface area allow the nonspecific bound contaminants to be very effectively removed from the column bed prior to elution.

Finally, fine control of the elution volume allows very small and complete elution of the purified protein producing the highest possible concentrations. Because the column is so highly loaded and the elution volume is small, the Virtual Heart-Cut Elution process produces a product as if the heart of a chromatography peak is recovered. This process produces concentrations that are 5-10 times higher than competing technologies. For example, 5 µL bed columns may employ elution volumes as low as 10 µL. Typically a single elution step will recover 60% of the bound protein while a follow-on second step will recover a further 20% of the bound protein. Finally, the separations can be easily run in parallel to develop the absolute superior conditions for purity, yield and concentration.

Many customers also use ELISA to test for protein activity. High protein recovery is useless unless the protein that is recovered has retained activity.

Four different antibody clones were screened for optimal purification conditions by varying 24 different buffer variables using PhyTip columns on a 96-channel robot. Yield was measured by ELISA and activity was measured by SPR.  In this way, purification conditions were screened and discovered that achieved both high recovery and high activity.

PHYTIP is a registered trademark of PhyNexus, Inc.

Protein Crystallography

X-ray crystallography is essentially a form of very high resolution microscopy.  It enables us to visualize protein structures at the atomic level and enhances our understanding of protein function.  Specifically we can study how proteins interact with other molecules, how they undergo conformational changes, and how they perform catalysis in the case of enzymes.  Armed with this information, we can design novel drugs that target a particular protein or rationally engineer an enzyme for a specific industrial process.

Crystallography Liquor Matrix Production – Lynx LM900 with the 96 VVP Pipetting Tool

VVP Residual Volume Data

The 96 VVP Pipetting Tool allows the ability to work from a worklist and aspirate from a deepwell plate with different crystallography liquors and then selectively dispense different volumes across multiple plates.

The system allows single or multiple dispenses from each tip over different wells defined in the database file. Using jet dispense mode, the head automatically increments of any of the wells to optimize combinations and dilutions to fit the crystallography application.

Crystallography Liquor Matrix Production – The Matrix Maker™ System
The Matrix Maker is a protein Crystallography liquid handling robot workstation that Dynamic Devices designed based on a collaborative relationship with Emerald BioStructures, Inc. a subsidiary of deCODE genetics http://decode.com. Currently, deCODE genetics is actively marketing the instrument in the proteomics marketplace for the use of protein crystallization in the field of structural biology. Dynamic Devices manufactures the complete liquid handling robot hardware system provides the low level software, custom components as well as support.

matrixmaker lg px600

The Matrix Maker™ system is a formulation preparation engine for high-throughput crystallography and formulations labs. Using Crystal Miner™ software, it solves the crystallization screen formulation bottleneck by allowing researchers to design and produce any desired reagent kit from up to 60 individual chemical stock solutions.

The liquid handling robot system is a unique combination of 30 precision syringe pumps, each with 9-position valve heads, innovative plumbing and a high-resolution gantry. It can accurately deliver chemical stock solutions under sterile conditions to any plasticware well or tube position.

The patented design allows the Matrix Maker to handle highly viscous solutions with excellent dispensing accuracy. All of the stock solutions are dispensed by positive pressure displacement, a design which eliminates aspiration problems and costly tip waste. From start to finish, the robot can produce a formulation matrix of 96 different cocktails in 35 minutes or less.

Automated MALDI Target Spotting

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maldi1

Either the Oasis LM 600 liquid handling robot or smaller Oasis CB 650 liquid handling robot platforms configured with the 96 channel low volume head make the perfect small footprint to spotting MALDI plates. The 96 head can transfer 0.5 µL form a microplate to each target site on the MALDI plate in either the 96 or 384 well format. Each plate has circular hydrophilic sample areas surrounded by hydroscopic areas allowing the samples to ‘stay’ in the required area since the plate is flat with no sides to hold in the liquid.

In addition, the 96 channel head can pick up matrixes of tips, like 6×6 or 4×4 tips for sample transfer to smaller format MALDI plates.