Microfluidic Dialysis Protein Crystallization Jiang Huang GN Biosystems, Inc. March 26, 2009
Dialysis Protein Crystallization Method Pro: scans a wide concentration range, the reagent composition can be easily altered during the course of the experiment. Con: difficult to setup, protein consumption too high (5 to 350 ml/rxn), not HT compatible.
Venn Diagrams Method Lysozyme VDX MB m. FD Hits 20 8 23 m. FD HANGING DROP 10 6 0 3 7 0 1 MICROBATCH Method Glucose isomerase VDX MB m. FD Hits 9 15 7 m. FD HANGING DROP 1 3 0 4 8 MICROBATCH Method Catalase VDX MB m. FD Hits 1 10 5 m. FD 0 HANGING DROP 2 3 0 7 MICROBATCH 1 0
Microfluidic Dialysis Plate - Design open bottom microtiter plate protein inlet film dialysis membrane discs microfluidic plate adhesive sealing tape
Microfluidic Dialysis Plate - Design dialysis membrane vacuum port protein inlet adhesive film reagent well
Microfluidic Dialysis Plate Design Top View Dialysis chambers dimensions: Screening plate: 18 nl per chamber (240 mm dia. , 400 mm deep). Optimization plate: 80 nl per chamber (500 mm dia. , 400 mm deep). Growth plate: 1 ml per chamber (1. 6 mm dia. , 400 mm deep) Bottom View Vacuum port Dialysis chamber Microfluidic channel Protein port
Microfluidic Dialysis Plate – Sample Loading
m. FD Method Highlights • Protein consumption as low as 15 nl per dialysis chamber • Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers • Low capital equipment costs to begin running experiments • High-throughput compatibility with standard dispensing robotics • Easy translation and scale-up designs • Chemically compatible with commercial reagent kits • The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation
m. FD Method Highlights • Protein consumption as low as 15 nl per dialysis chamber • Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers • Low capital equipment costs to begin running experiments • High-throughput compatibility with standard dispensing robotics • Easy translation and scale-up designs • Chemically compatible with commercial reagent kits • The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation
m. FD Method Highlights • Protein consumption as low as 15 nl per dialysis chamber • Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers • Low capital equipment costs to begin running experiments • High-throughput compatibility with standard dispensing robotics • Easy translation and scale-up designs • Chemically compatible with commercial reagent kits • The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation
Equipment and Accessories Needed 1. Vacuum pump or house vacuum with an ultimate vacuum ≤ 0. 1 mm. Hg *Air bubbles in every dialysis chamber will result due to insufficient vacuum A List of Qualified Vacuum Pumps at under $2, 000 Manufacture Model# Ultimate Vacuum BOC/Edwards Welch Brinkmann Thermo-Electron RV 3 EVA 480 -16 -941 1400 B-01 1399 B-01 V-500 3178712 3178707 1× 10 -6 mm. Hg 8× 10 -3 mm. Hg 1× 10 -4 mm. Hg 0. 02 mm. Hg 0. 01 mm. Hg 3. 8× 10 -3 mm. Hg 1× 10 -4 mm. Hg
m. FD Method Highlights • Protein consumption as low as 15 nl per dialysis chamber • Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers • Low capital equipment costs to begin running experiments • High-throughput compatibility with standard dispensing robotics • Easy translation and scale-up designs • Chemically compatible with commercial reagent kits • The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation
m. FD Method Highlights • Protein consumption as low as 15 nl per dialysis chamber • Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers • Low capital equipment costs to begin running experiments • High-throughput compatibility with standard dispensing robotics • Easy translation and scale-up designs • Chemically compatible with commercial reagent kits • The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation
Translation and Scale-up screening dia. =240 mm depth=400 mm optimization dia. =500 mm depth=400 mm growth dia. =1. 6 mm depth=400 mm 50 mg/ml Lysozyme vs. HCS 1 #10 via diameter: 1. 2 mm (570 nl volume) crystal size: up to 500 mm long via diameter: 0. 3 mm (20 nl volume) crystal size: up to 50 mm long
Translation and Scale-up Hampton Crystal Screen I 15 ml dialysis bottom #2 Optimization Plate 20 x Screening Plate 90 x #6 #7 #9 #13 #14
m. FD Method Highlights • Protein consumption as low as 15 nl per dialysis chamber • Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers • Low capital equipment costs to begin running experiments • High-throughput compatibility with standard dispensing robotics • Easy translation and scale-up designs • Chemically compatible with commercial reagent kits • The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation
m. FD Method Highlights • Chemically compatible with commercial reagent kits (materials used: PMMA, epoxy, dialysis membrane) M. W. Toepke, D. J. Beebe, PDMS absorption of small molecules and consequences in microfluidic applications, Lab Chip, 2006, 6: 1484 -1486
m. FD Method Highlights • Protein consumption as low as 15 nl per dialysis chamber • Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers • Low capital equipment costs to begin running experiments • High-throughput compatibility with standard dispensing robotics • Easy translation and scale-up designs • Chemically compatible with commercial reagent kits • The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation
Microfluidic Dialysis Plate - Design dialysis membrane vacuum port protein inlet adhesive film reagent well
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