docPhyTip® column technology
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PhyTip® column technology
Two application highlights: The AutoPlasmid MEA and Gel Filtration Lee Hoang PhyNexus, Inc. San Jose, CA CAPTURE PURIFY ENRICH™ PhyNexus PhyTip® column design Top screen Resin maintained in discrete area Bottom screen CAPTURE PURIFY ENRICH™ PhyNexus PhyTip® column design Affinity PhyTip Columns 5 20 10 20 40 80 Gel Filtration 160 320 mL 200 mL 600 mL CAPTURE PURIFY ENRICH™ Plasmid 80 µL PhyNexus AutoPlasmid MEA CAPTURE PURIFY ENRICH™ PhyNexus Processes requiring high-throughput plasmid preps Affinity maturation Protein-protein interactions Comprehensive yeast-2-hybrid Structural genomics Protein microarrays Expression Screening DNA and cDNA libraries CAPTURE PURIFY ENRICH™ PhyNexus The major challenge to current technologies CAPTURE PURIFY ENRICH™ PhyNexus PhyNexus Solution CAPTURE PURIFY ENRICH™ PhyNexus AutoPlasmid MEA For complete automation of mini, midi and maxi DNA preps CAPTURE PURIFY ENRICH™ PhyNexus AutoPlasmid MEA For complete automation of mini, midi and maxi DNA preps Includes Software Methods for Ease of Use Default methods for: • Miniprep • Miniprep of low copy number plasmids • Midiprep 5 mL culture • Midiprep 20 mL culture • Maxiprep 100 mL culture CAPTURE PURIFY ENRICH™ PhyNexus AutoPlasmid MEA For complete automation of mini, midi and maxi DNA preps Includes Software Methods for Ease of Use Tune Bacteria Culture Conditions to Meet Yield Needs • Plasmid copy number • Medium • Culture Vessel CAPTURE PURIFY ENRICH™ PhyNexus DNA Miniprep Application Two methods in place for up to 48 samples/run • Medium and high copy number plasmids • Low copy number plasmids Kit sold as single box or multi-box. Buffers scaled up as necessary. CAPTURE PURIFY ENRICH™ PhyNexus DNA Midiprep Application Two methods for yields of 50 µg • 5 mL cultures, 36 samples/run • 5+ mL cultures, 24 samples/run Use 4x6 plates and larger buffer volumes CAPTURE PURIFY ENRICH™ PhyNexus DNA Maxiprep Application Perform Midiprep with multiple PhyTip columns and pool for yields up to 1 mg 2-3 samples/run CAPTURE PURIFY ENRICH™ PhyNexus Perfornmance Specifications Complete Automation • Multi-sample format • Walk-away automated • Capable of high-throughput • No hands-on intervention Compatible with Downstream Assays • DNA sequencing • PCR • Restriction enzyme digestion • Transformation/Transfection Purify Plasmid from E. coli Lysate • Tune culture for yield •No RNA contamination • A260/A280 ratio between 1.8-2.0 • A260/A230 ratio between 2.0-2.2 Streamline • Just one centrifugation step • No additional accessories CAPTURE PURIFY ENRICH™ PhyNexus Gel Filtration CAPTURE PURIFY ENRICH™ PhyNexus A gel filtration column contains beads with pores and channels running through them 1. Small molecules penetrate the beads and travel into the channels 2. Large molecules are excluded from the channels. CAPTURE PURIFY ENRICH™ PhyNexus CAPTURE PURIFY ENRICH™ PhyNexus CAPTURE PURIFY ENRICH™ PhyNexus CAPTURE PURIFY ENRICH™ PhyNexus CAPTURE PURIFY ENRICH™ PhyNexus CAPTURE PURIFY ENRICH™ PhyNexus CAPTURE PURIFY ENRICH™ PhyNexus CAPTURE PURIFY ENRICH™ PhyNexus CAPTURE PURIFY ENRICH™ PhyNexus CAPTURE PURIFY ENRICH™ PhyNexus CAPTURE PURIFY ENRICH™ PhyNexus Proteins are loaded onto the column. CAPTURE PURIFY ENRICH™ PhyNexus Smaller molecules enter into the pores and channels. These molecules take longer to elute. CAPTURE PURIFY ENRICH™ PhyNexus Larger molecules are excluded from the pores and channels. These molecules move between the beads and elute first. CAPTURE PURIFY ENRICH™ PhyNexus Larger molecules are excluded from the pores and channels. These molecules move between the beads and elute first. CAPTURE PURIFY ENRICH™ PhyNexus Larger molecules are excluded from the pores and channels. These molecules move between the beads and elute first. CAPTURE PURIFY ENRICH™ PhyNexus Larger molecules are excluded from the pores and channels. These molecules move between the beads and elute first. CAPTURE PURIFY ENRICH™ PhyNexus Gel Filtration with PhyTip Columns 1 1 2 3 4 5 6 7 • Routinely obtain 80% protein recovery & remove >95% salt • Separation of myoglobin (brown MW 16.7kD) from DNP-glutamate (yellow, MW 313D) • Elution fractions collected in separate wells 2 3 4 5 6 7 Tips from left: Tubes from left: 1. 2. 3-6. 7. 1. Starting sample 2. 200 µL fraction 3-6. 100 µL fractions 7. 400 µL fraction 600 µL column 200 µL sample load 4x100 µL chaser 400 µL chaser CAPTURE PURIFY ENRICH™ PhyNexus Compatible with small volume samples Suggested sample and elution volumes Column bed size (mL) 200 200 200 200 200 200 200 200 600 600 600 600 Sample vol. (mL) 20 30 40 50 60 70 80 90 100 200 300 400 Recommended chaser vol. (mL) 150 140 130 120 110 100 90 80 400 300 200 100 CAPTURE PURIFY ENRICH™ PhyNexus Buffer exchange and desalting IgG recovery studies For separation and removal of excess dye, biotinylation reagents, and buffer exchange Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Ave SD CV Final Volume (mL) 122 119 122 119 122 123 121 2 1.4 Concentration (mg/mL) 0.49 0.50 0.50 0.54 0.48 0.51 0.50 0.02 4.1 Mass Recovered (mg) 0.060 0.060 0.061 0.064 0.059 0.063 0.061 0.002 3.5 CAPTURE PURIFY ENRICH™ % Protein Recovered 75 74 76 80 73 78 76 3 4 PhyNexus Buffer exchange and desalting Separation and removal of imidazole after IMAC PhyTip column purification Sample Pre-Desalting His-Ubiquitin Sample Post-Desalting Blank CAPTURE PURIFY ENRICH™ PhyNexus PhyTip Gel Filtration Adaptor for 96-channel Liquid Handling CAPTURE PURIFY ENRICH™ PhyNexus The PhyNexus Solution • Pipette tip-based purification column technology compatible with most automation platforms • Enables miniaturization of all affinity and solid-phase chromatography chemistries • High yield, gentle on protein, small elution volume • 1 - 96 samples in an automated platform for rapid screening • Results are reproducible, scalable and transferable CAPTURE PURIFY ENRICH™ PhyNexus