Using the TOPAZ 4.96 Chip

Transcription

1 Using the TOPAZ 4.96 Chip On the TOPAZ FID Crystallizer P/N , Rev. B September, 2004

2 Fluidigm Corporation and its suppliers own the written and other materials in the TOPAZ FID Crystallizer. The written materials are copyrighted and protected by the copyright laws of the United States of America and, throughout the world, by the copyright laws of each jurisdiction according to pertinent treaties and conventions. No part of this manual may be reproduced manually, electronically, or in any other way without the written permission of Fluidigm Corporation. Fluidigm is a registered trademark of Fluidigm Corporation. OptiMix, MSL, and TOPAZ are trademarks of Fluidigm Corporation. All of the trademarks, service marks, and logos used in these materials are the property of Fluidigm Corporation or their respective owners. You may not use, and nothing contained in these materials grants by implication, waiver, estoppel or otherwise, any right in any trademark displayed in these materials without the written permission of Fluidigm Corporation or the respective owner. NOTICE TO PURCHASER: For Research Use Only. Not for use in diagnostic procedures. Patents pending. Copyright Fluidigm Corporation. All rights reserved. Made in the USA Contacting Fluidigm For Technical Support, send to: TechSupport@fluidigm.com Phone in United States: FLUIDLINE ( ) Outside the United States: On the Internet: Fluidigm Corporation 7100 Shoreline Court South San Francisco, CA 94080

3 CONTENTS CONTENTS Introducing the 4.96 Chip Features of the 4.96 Chip Handling Reagents and Proteins Setting Up a Chip Run on the 4.96 Chip Critical Timing Requirements for Setup Staggering Chip Preparation Steps FID Crystallizer Software Scripts for the 4.96 Chip Preparation of the 4.96 Chip Delivery of Reagents Dispensing Protein into the Protein Inlet Pressurizing Reagent and Protein Delivery Initiating Diffusion Incubating Chips Inspecting for Crystals Troubleshooting Ordering Additional Parts INDEX Fluidigm Corporation, 2004

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5 I NTRODUCING THE 4.96 CHIP The 4.96 Chip supports a crystallization screen of 96 reagents against up to four proteins or samples. Incorporating an integrated fluidic circuit (IFC), reagent inlets, four protein inlets, and two accumulators in one disposable unit, the 4.96 Chip conforms to microplate dimensions, making it compatible with devices for automated handling and liquid dispensing. The 4.5 mm grid spacing between the centers of the 96 reagent inlets is also compatible with multichannel pipettors Figure 1 The 4.96 Chip with hydration chamber and PVA strips in reservoirs. # Component # Component 1 Notched corner (Locator for A1 on an SBS 384-well plate) 2 Containment accumulator check valve 3 Barcode label on side (not visible in this figure) 5 Interface accumulator check valve 6 Protein inlets (equivalent to A11 A14 on an SBS 384-well plate) 7 Integrated fluidic circuit (IFC) 4 Reagent inlets 8 Hydration chamber, with lid, and PVA strips in outer reservoirs 1

6 Introducing the 4.96 Chip Features of the 4.96 Chip When introduced into the chip s reagent inlets, the 96 reagents are distributed into 384 diffusion experiments on the elastomer IFC in the 4.96 Chip. In all diffusion experiments of the 4.96 Chip, the ratio of reagent to protein is 3.4:1. Notched Corner at A1 The notched corner of the 4.96 Chip the corner closest to position A1 in the numbering grid used on an SBS 384-well plate fits into the corner at the front of each controller bay, labeled A1. A light sensor at each bay detects when the 4.96 Chip is oriented and seated properly. Use the notched corner as a guide when placing the 4.96 Chip on the controller and when pipetting reagents. IMPORTANT Correct orientation of the notched corner on the 4.96 Chip is critical to assure pressurized delivery of protein and control line fluid! Always align the notched corner of the 4.96 Chip with the corner of the controller bay that is labeled A1. Protein Inlets When the notched corner at A1 is at the top left corner, the protein inlets are the four inlets above the hydration chamber. (See Figure 1 on page 1.) Four blue squares on the side of the chip label the protein inlets. Only fill these inlets with protein. Hydration Chamber The IFC in the center of the 4.96 Chip is surrounded by a hydration chamber. Sterile, distilled deionized water, combined with Hydration Fluid, is applied to two strips of polyvinyl alcohol (PVA) polymer in the hydration chamber to hydrate the 4.96 Chip before setting up experiments. A lid covers the entire hydration chamber to maintain chip environment. 2

7 Features of the 4.96 Chip Accumulators The accumulators currently maintain pressure to hold the corresponding control channel valves closed during incubation for at least 7 days. Each accumulator has a check valve that, when depressed, discharges accumulated pressure to open the associated control valves. Barcode Label A barcode label on the outside edge of the containment accumulator displays each chip s unique identification number. Control Channels Two layers of elastomer are bonded to the substrate of the 4.96 Chip. The top elastomer layer, or control layer, contains two control channels; the bottom elastomer layer, or fluid layer, contains protein and reagent channels. The control channels consist of interface and containment channels. As shown in Figure 2 on page 4, the interface channel separates the protein and reagent chambers; pressure to the interface accumulator controls the interface valves. The containment valves, which isolate each diffusion experiment during incubation, are controlled by pressure from the containment accumulator. During incubation, the interface channel can also be repressurized to control diffusion of reagent into the protein chamber and maintain protein concentration. 3

8 Introducing the 4.96 Chip Reagent chambers Interface valve Containment valve Protein chambers Figure 2 Diagram of eight diffusion experiments in a 4.96 Chip. Diffusion Experiments and Cells A diffusion experiment consists of a pair of chambers one for protein and one for reagent separated by an interface valve, and extends to the containment valves on either side of the chambers (Figure 2). With four protein inlets and 96 reagent inlets on the 4.96 Chip. The ratio of reagent to protein in each diffusion experiment is 3.4:1, with 2.5 nl reagent and 0.73 nl protein. During the setup of a crystallization experiment on the 4.96 Chip, the interface valve is closed while each chamber in a diffusion experiment is filled with either reagent or protein. After the chambers are filled, the containment valves are closed to create 384 unique diffusion experiments. When the interface valve is opened, diffusion of the protein and reagent begins. After sufficient time for equilibration has passed, the interface valve can be closed to terminate free interface diffusion (FID). Numbering of cells and diffusion experiments The 96 diffusion experiments are distributed into 4 columns of 24 cells labeled A-1 through A-24, B-1 through B24, C1 through C24, and D1 through D24 that are located in the elastomer IFC in the center of the 4.96 Chip. Each cell contains 4 diffusion experiments. 4

9 Features of the 4.96 Chip Figure 3 on page 6 shows the numbering system for the cells, which is visible on the 4.96 Chip, and the corresponding diffusion experiments associated with each cell. The diffusion experiment numbers are not embedded in the elastomer IFC. The number assigned to one diffusion experiment is a combination of two other numbers: the number of a protein inlet plus the number of a reagent inlet. For example, the reagent that is pipetted into reagent inlet 3 moves into four diffusion experiments 1.03, 2.03, 3.03, and Each of these diffusion experiments contains protein from a different protein inlet. As shown in Figure 3, these diffusion experiments are located in two different cells D02 and B02. 5

10 Introducing the 4.96 Chip Two adjacent cells on the 4.96 Chip. Each cell is a locator for four diffusion experiments. Diffusion experiments 4.03, 3.03, 4.07, and 3.07 are located in cell B02. Diffusion experiment 4.03 contains: protein from protein inlet 4 reagent from reagent inlet 3 Figure 3 Cell numbers and associated diffusion experiments on the elastomer IFC in the center of the 4.96 Chip. 6

11 Features of the 4.96 Chip Reagent and Protein Inlets The 4.96 Chip has 96 reagent inlets and four protein inlets. Reagents are pipetted into six columns of sixteen inlets located on either side of the IFC. The four protein input inlets are located at the equivalent to position A11 A14 in a 384-well grid. Figure 4 is an illustration of the layout of cells, reagent inlets, and protein inlets on the 4.96 Chip and their numbering systems. Reagent inlets Protein inlets Reagent inlets Cell numbers Figure 4 Numbering systems for protein inlets, reagent inlets, and cells on the 4.96 Chip. See Figure 3 on page 6 for a map of diffusion experiments on the central IFC. 7

12 Introducing the 4.96 Chip Handling Reagents and Proteins It is crucial that you keep your experiments free of contamination. Proteins are susceptible to microbial contamination and damage from proteases. Contamination of the TOPAZ Chip or reagents can affect crystallization or confuse interpretation if it appears in the wells and channels of the elastomer IFC. IMPORTANT To prevent contamination, wear powder-free gloves when working with the TOPAZ Chip, protein, and reagents. Proper preparation is critical to successful protein crystallography experiments. Use the following guidelines to prepare your protein: Filter or centrifuge the protein before use to remove any aggregates or particulates that can clog the fluid channels. Avoid introducing air bubbles into the protein solution. IMPORTANT When using centrifugation to prepare small aliquots, take extreme care to avoid dislodging the pellet with the pipette tip and accidentally introducing aggregated particulates into the IFC. Always use sterile buffer solutions. Store the protein at or below 4 C and minimize the amount of time it is left at room temperature to avoid microbial contamination. Always use sterile buffer solutions. Store the protein at or below 4 C and minimize the amount of time it is left at room temperature to avoid microbial contamination. Use freshly-made protein whenever possible. If protein must be frozen, store proteins in small aliquots (10 20 µl) at 80 C to avoid freeze-thaw cycles. Slowly equilibrate the protein solution to the temperature at which the experiment will be carried out prior to setting up an experiment with the TOPAZ Chip. If your sample contains unpaired cysteine residues, add fresh reducing agent (10 mm dithiothreitol [DTT]; % Tri [2- carboxyethyl]phosphine hydrochloride [TCEP], ph 7; or 20 mm β-mercaptoethanol) to your sample immediately prior to 8

13 Handling Reagents and Proteins setting up crystallization experiments. If you believe that your protein is particularly sensitive to oxidation, reducing agent should also be added to the crystallization reagents. Quantify protein concentration before setting up chip runs to ensure reproducibility for repeat experiments. We recommend that the concentration be measured using absorbance at 280 nm and an extinction coefficient at 280 nm for your protein, calculated from the protein sequence. If your protein solution contains cofactors, compounds, ligands, or other additives that absorb significantly at 280 nm, we recommend using a colorimetric estimation of protein concentration such as the BCA Protein Assay (Pierce Biotechnology), or the Coomassie protein assay (Bradford, 1976). Lyophilization may inhibit protein crystallization and should be avoided if possible. If lyophilization cannot be avoided, dialyze the protein prior to use in the TOPAZ Chip. It may also be necessary to dialyze, centrifuge, or filter the protein prior to metering it into the TOPAZ Chip to remove unwanted buffers or chemicals, microbes, or sample aggregation. 9

14 Introducing the 4.96 Chip 10

15 S ETTING UP A CHIP RUN ON THE 4.96 CHIP This chapter describes how to use the scripts in FID Crystallizer software to set up a chip run on the FID Crystallizer with a 4.96 Chip. The steps to setting up a chip run on the 4.96 Chip are: 1 Hydrate the 4.96 Chip. See page Run the 1 Prep script to activate control channels. See page Pipette reagents and protein. See page 19 and page Run the 2 Load 4.96 script to pressurize delivery of reagents and protein. See page Perform T0 scan on the TOPAZ AutoInspeX Workstation. See page Run 3 FID Control RT or 3 FID Control 4C to initiate controlled diffusion of reagent and protein. See page Inspect for crystals and recharge the accumulator during incubation. See page

16 Setting Up a Chip Run on the 4.96 Chip Critical Timing Requirements for Setup Throughout the setup of diffusion experiments on the 4.96 Chip, the following timing requirements must be observed to assure optimum chip performance. Table 1 Steps with Critical Timing Requirements Step Perform... See... Add diluted Hydration Fluid to the chip and reseat lid. Run the 1 Prep script. Dispense protein to chip Run the 2 Load 4.96 script Perform T0 scan (optional) immediately after package is opened within 20 hr. after adding diluted Hydration Fluid within 60 min. after reagent dispensing within 5 min. after protein dispensing within 60 min. after loading script is finished page 17 page 19 page 20 page 23 page 24 page 24 Run the 3 FID Control RT or 3 FID Control 4C script within 60 min. after T0 scan page 27 Run the 7 Recharge script within 7 days after incubation begins page 28 IMPORTANT Delivery of pressurized gas to the FID Crystallizer controller continues throughout the duration of any of the scripts listed in Table 2 on page 14. In laboratories that plumb compressed gas tanks to the FID Crystallizer controller, we recommend use of the 3 FID Start script in conjunction with the 4 FID End script to conserve compressed gas during controlled diffusion. Staggering Chip Preparation Steps Once you become comfortable with the steps described in this chapter, with the aid of a robot for reagent dispensing you can stagger the steps to set up chip runs on eight chips in approximately 4.5 hours, as shown in Figure 5 on page

17 Staggering Chip Preparation Steps Without the aid of a robot, you may follow a similar staggered sequence of steps. Allow additional time for reagent pipetting. START Prepare four 4.96 Chips: Add diluted Hydration Fluid (page 17) Run 1 Prep script (page 19) Dispense reagents via robot (page 20) Dispense proteins (page 23) Run 2 Load 4.96 script (page 24) Scan chips on TOPAZ AutoInspex Workstation (page 24) Prepare four 4.96 Chips: Add diluted Hydration Fluid (page 17) Run 1 Prep script (page 19) Dispense reagents via robot (page 20) Dispense proteins (page 23) Run 2 Load 4.96 script (page 24) Run 3 FID Start (page 27) Scan chips on TOPAZ AutoInspex Workstation (page 24) Run 4 FID End (page 28) Run 3 FID Start (page 27) INCUBATION Run 4 FID End (page 28) (Run 7 Recharge at day 7 to recharge accumulators.) Figure 5 Staggering protocol steps for setting up chip runs at room temperature with 4.96 Chips. By staggering steps, a skilled user can set up 8 chip runs (3072 diffusion experiments) in approximately 4.5 hours. 13

18 Setting Up a Chip Run on the 4.96 Chip IMPORTANT Always wear proper laboratory clothing protective gloves, lab coat, safety glasses when handling any chemicals, reagents, or TOPAZ Chips. FID Crystallizer Software Scripts for the 4.96 Chip Table 2 lists the scripts that are available in FID Crystallizer software for use with the 4.96 Chip. Table 2 Scripts for the 4.96 Chip Script Name Time Description 1 Prep 1 min. Activates control valves. Use to prepare 4.96 Chip for chip run. 2 Load min. Closes containment valves, pressurizes reagent and protein blindfill up to containment valves, closes interface valves, opens containment valves, continues loading protein and reagent up to interface valve, closes containment valve. Use after 1 Prep to load reagents and protein. Chip is ready for T0 scan at end of script. 3 FID Control 4C 100 min. Opens interface valves to begin diffusion. After a 100-min. period of diffusion, closes interface valves and recharges containment accumulator. Default recommended script for use at 4ºC, after T0 scan. As an alternative, use 3 FID Start to begin diffusion and 4 Recharge to interrupt FID after 100 min. 3 FID Control RT 60.5 min. Opens interface valves to begin diffusion. After a 60-min. period of diffusion, closes interface valves and recharges containment accumulator. Default recommended script for use at room temperature, after T0 scan. As an alternative, use 3 FID Start to begin diffusion with 4 Recharge to interrupt FID after 60 min. 14

19 FID Crystallizer Software Scripts for the 4.96 Chip Script Name Time Description 3 FID Start 30 sec. Opens interface valves to begin diffusion. Use after T0 scan. as an alternative to either 3 FID Control RT or 3 FID Control 4C. Follow after recommended diffusion time with the 3 FID End script. See Table 6 on page 28 for recommended diffusion times. 4 FID End 30 sec. Closes interface valves to end diffusion. Use in conjunction with 3 FID Start. 5 Auto RT min. Merges 1 Prep, 2 Load 4.96, and 3 FID Control RT. Use at room temperature to prepare, blind fill reagents and protein, begin diffusion, and then stop FID after 60 min. After initial preparation, contains a pause step to allow the introduction of reagents and protein. After blind fill, contains a pause to verify chambers completely filled. Use if T0 scan is not needed. 5 Auto 4C min. Merges 1 Prep, 2 Load 4.96, and 3 FID Control 4C. Use at 4 ºC to prep, blind fill reagents and protein, begin diffusion and then stop FID after 100 min. After initial preparation, contains a pause step to allow the introduction of reagents and protein. After blind fill, contains a pause to verify chambers completely filled. Use if T0 scan is not needed. 6 Reload min. Closes interface valves, opens containment valves, pressurizes reagent and protein blindfill up to interface valves, closes containment valves. Use if chambers are not completely filled at the end of the 2 Load 4.96 script. Chip is ready for T0 scan at end of script. 7 Recharge 30 sec. Recharges interface and containment accumulator. Use every 7 days during incubation. 15

20 Setting Up a Chip Run on the 4.96 Chip IMPORTANT Delivery of pressurized gas to the FID Crystallizer controller continues throughout the duration of any of the scripts listed in Table 2 on page 14. In laboratories that plumb compressed gas tanks to the FID Crystallizer controller, we recommend use of the 3 FID Start script in conjunction with the 3 FID End script to conserve compressed gas during diffusion on the chip. Preparation of the 4.96 Chip In preparation for shipment, the 4.96 Chip is prehydrated to 95% relative humidity (RH) and sealed in a package with humidity packs. When you remove the chip from its packaging, you must pipette diluted Hydration Fluid onto the strips of polyvinyl alcohol (PVA) polymer on either side of the chip and replace the lid immediately after opening the package to maintain humidity. IMPORTANT Before you open the chip packaging, have sufficient volume of diluted Hydration Fluid ready to introduce onto the PVA strips. Hydration Fluid solutions The dilution of Hydration Fluid Concentrate required for your chip run depends on the OptiMix Reagent Kit you are using. See Table 3. Each 4.96 Chip requires 1.5 ml of diluted Hydration Fluid. Prepare enough diluted Hydration Fluid for all the chips you will use in a day. To prepare Hydration Fluid For a 2:1 (v/v) solution, add 2 parts sterile D.I. water to 1 part Hydration Fluid Concentrate. For 1:1 (v/v) solution, add 1 part sterile D.I. water to 1 part Hydration Fluid Concentrate. 16

21 Preparation of the 4.96 Chip Table 3 Hydration Fluid Dilution Matrix OptiMix Reagent Kit Chip Humidity Hydration Fluid Dilution (v/v) OptiMix-1 90% 2:1 OptiMix-2 90% 2:1 OptiMix-3 80% 1:1 OptiMix-PEG 90% 2:1 To add Hydration Fluid to the TOPAZ Chip 1 Remove the 4.96 Chip from its packaging and raise the lid that covers the hydration chamber. 2 Immediately add 750 µl of the appropriate Hydration Fluid dilution to each strip of PVA in the hydration chamber reservoirs. See Figure 6 on page 18. Note Make sure the PVA strips lie in the outer reservoirs, away from the central IFC. Do not allow diluted Hydration Fluid to make contact with the IFC. 3 Immediately replace the hydration chamber lid and press the tab firmly into place. The lid should be flat and the edges of the lid should be completely seated within the walls of the hydration chamber. The PVA absorbs the liquid within minutes. Note To maintain humidity, keep the lid on the hydration chamber throughout chip run and chip scanning. 17

22 Setting Up a Chip Run on the 4.96 Chip Figure 6 The 4.96 Chip # Component # Component 1 Notched corner (A1) 4 Protein inlets (1 4) 2 Check valve, containment accumulator 5 Check valve, interface accumulator 3 PVA strip in outer reservoir 6 Hydration chamber with integrated fluidic circuit (IFC) IMPORTANT Activate control valves and dispense reagents within 20 hr. of adding diluted Hydration Fluid. IMPORTANT Do not place labels on the top or the bottom of the 4.96 Chip where they may hinder the seal between the chip and the gasket on the controller bay. You may place a label on the side of the chip, but it should lay flush and should not fold over onto the top of the chip or onto the protruding flange at the bottom. 18

23 Delivery of Reagents Activating Control Valves on the 4.96 Chip Run the 1 Prep script to activate control valves. Note With Group Mode, you can run the same script simultaneously at two or more controller bays. Refer to the TOPAZ FID Crystallizer User Guide for information about using the features available in FID Crystallizer software. To activate the control valves 1 In the FID Crystallizer software, enter the chip and chip run information at a bay position. Scan or enter the chip Barcode. Enter Experiment name. Enter the Operator name Select 1 Prep script. 2 Place the prepared 4.96 Chip on the corresponding FID Crystallizer controller bay. Align the notched corner of the chip with the A1 label in the bay. 3 Press Start. 4 When the script is completed, click Finish. IMPORTANT Reagents must be dispensed within 20 hr. of chip priming. Delivery of Reagents On the 4.96 Chip, the 96 reagent inlets are distributed across 6 columns of 16 inlets, located on either side of the elastomer IFC, as shown in Figure 7 on page 21. The reagent inlets are numbered 1 through 96, with 1 in the top left corner and 96 in the bottom right corner. 19

24 Setting Up a Chip Run on the 4.96 Chip IMPORTANT The 4.96 Chip is compatible with aqueous solutions in the range of ph Use of organic solvents at concentrations beyond those contained in commercial screens is not recommended. We strongly recommend the use of OptiMix Reagents with TOPAZ Chips. To dispense reagents to the 4.96 Chip Use a 20 µl pipettor, multi-channel pipettor, or liquid-dispensing robot to inject at least 10 µl reagent into each reagent inlet. IMPORTANT When pipetting manually, make sure there are no air bubbles in the pipette tip. Pipette to the bottom of each inlet. Press down only to the first pipettor stop to avoid introducing air into the bottom of the inlet. When using a liquid-dispensing robot, set the height of the pipette tips as close as possible to the bottom of each reagent inlet to minimize the formation of air bubbles. When pipetting viscous reagents, allow sufficient time for complete delivery. IMPORTANT Protein must be dispensed within 60 min. of reagent dispensing. Dispensing reagents with an eight-channel pipette When using an eight-channel pipette to dispense reagents from a 96-well block that has eight columns A through H of reagent wells numbered 1 through 12. The pipette tips disperse to every other reagent inlet in the vertical column of inlets on the 4.96 Chip. Reagent is dispersed in rows, from left to right, as shown in Figure 7. 20

25 Delivery of Reagents A1 corner Reagent inlets Reagent inlets 1 96 Figure 7 Pattern of reagent inlets on the 4.96 Chip. Dotted lines show the two alternating horizontal paths of an 8-channel pipettor dispensing to the reagent inlets. Table 4 on page 22 shows how the numbers of the wells in an OptiMix Reagent Kit plate (96-wells) correspond to the numbers of the reagent inlets on the 4.96 Chip when using a multi-channel pipettor to pipette reagents into the chip. Table 4 assumes that reagents are dispensed from left to right, in two alternating horizontal paths, as shown in Figure 7. 21

26 Setting Up a Chip Run on the 4.96 Chip Table 4 Numbering Systems on 96-well Reagent Plate vs Chip Plate Well Chip Inlet Plate Well Chip Inlet Plate Well Chip Inlet Plate Well Chip Inlet Plate Well Chip Inlet Plate Well Chip Inlet A-1 1 A-2 2 A-3 3 A-4 4 A-5 5 A-6 6 A-7 7 A-8 8 A-9 9 A A A B-1 13 B-2 14 B-3 15 B-4 16 B-5 17 B-6 18 B-7 19 B-8 20 B-9 21 B B B C-1 25 C-2 26 C-3 27 C-4 28 C-5 29 C-6 30 C-7 31 C-8 32 C-9 33 C C C D-1 37 D-2 38 D-3 39 D-4 40 D-5 41 D-6 42 D-7 43 D-8 44 D-9 45 D D D E-1 49 E-2 50 E-3 51 E-4 52 E-5 53 E-6 54 E-7 55 E-8 56 E-9 57 E E E F-1 61 F-2 62 F-3 63 F-4 64 F-5 65 F-6 66 F-7 67 F-8 68 F-9 69 F F F G-1 73 G-2 74 G-3 75 G-4 76 G-5 77 G-6 78 G-7 79 G-8 80 G-9 81 G G G H-1 85 H-2 86 H-3 87 H-4 88 H-5 89 H-6 90 H-7 91 H-8 92 H-9 93 H H H Dispensing with the aid of a robot When applying the numbering grid used on an SBS 384-well plate, reagents on the left side of the 4.96 Chip are in wells A 4, A5, A6 through P4, P5, P6. On the right side of the 4.96 Chip, reagents are in wells A19, A20, A21 through P19, P20, P21. When applying the numbering grid used on an SBS 384-well plate, protein inlets 1 through 4 are on the 4.96 Chip is at positions A11 through A14. The notched corner of the 4.96 Chip is at position A1. See also Appendix B in the TOPAZ FID Crystallizer User Guide for recommended specifications for liquid handling robots. 22

27 Dispensing Protein into the Protein Inlet Dispensing Protein into the Protein Inlet The four protein inlets, numbered 1 4, are at the top of the 4.96 Chip. IMPORTANT There are 4 protein inlets across the top of the chip. Refer to Figure 6 on page 18 for the location of the protein inlets. Protein Dispensing Guidelines When pipetting microliter amounts of protein, take the following precautions to assure adequate delivery of protein into the protein inlet: Use a well-calibrated pipettor. Make sure there are no air bubbles in the pipette tip. Pipette directly to the center of the bottom of the protein inlet. Avoid pipetting to the inlet sides. Before depressing the plunger, make sure the pipette tip is level and at the bottom of the protein well. Depress the plunger to the first stop and wait one second before removing the tip. After removing the pipette tip from the inlet, verify that the tip is empty and that protein has been delivered into the inlet. To dispense protein Use a well-calibrated pipettor and a P10 or gel-loading pipette tip to pipette at least 1.4 µl protein into the bottom of each protein inlet (positions A11 through A14 on an SBS 384-well grid). IMPORTANT After protein is dispensed to the protein inlets, begin pressurized loading of the 4.96 Chip within 5 minutes to minimize loss of protein from evaporation. 23

28 Setting Up a Chip Run on the 4.96 Chip Pressurizing Reagent and Protein Delivery Use the 2 Load 4.96 script to pressurize delivery of reagents and protein into diffusion chambers on the 4.96 Chip at all temperatures. Note See page 14 for scripts that combine prep, blindfill, and controlled FID at room temperatures and 4 ºC. You may use these scripts if you do not perform a chip scan at T0. To pressurize reagent and protein delivery 1 In the FID Crystallizer software, enter the chip barcode and operator name at a bay position. The chip barcode is linked to the Experiment name that was previously entered. 2 Select the 2 Load 4.96 script. 3 Place the prepared 4.96 Chip on the corresponding FID Crystallizer controller bay. 4 Press Start. 5 At the end of the loading script, click Finish. If loading is not complete at the end of 2 Load 4.96, run 6 Reload 4.96 to finish loading. If you used a 5Auto... script, the chip is now ready for incubation. See page 25. IMPORTANT Perform a T0 scan within 60 min. and initiate FID within 120 min. of pressurized reagent and protein loading. Scanning at T0 At the end of the loading script for the 4.96 Chip, when pressurized delivery of reagents and protein is complete, you can use the TOPAZ AutoInspeX Workstation to perform a Time=Zero (T0) scan. At T0, all chambers should be filled with reagents or protein and the interface and containment valves should be closed, as shown in Figure 8 24

29 Initiating Diffusion on page 25. Figure 9 shows a reagent chamber that is not completely filled. Protein chambers Reagent chambers Closed containment valves Closed interface valve Figure 8 Reagent and protein chambers are completely filled, containment and interface valves are closed at T0. Green and yellow circles highlight examples of closed valves. Protein chambers Reagent chambers Closed interface valve Closed containment valves Figure 9 An example of a reagent chamber that is not completely filled. If loading is not complete at the end of 2 Load 4.96, run 6 Reload 4.96 to finish loading If all chambers are not filled at the end of either loading script, see page i. Initiating Diffusion The 4.96 Chip allows crystallization to occur through a combination of free interface diffusion (FID) and dehydration. A lid in the center of the 4.96 Chip covers the entire IFC, creating a closed hydration chamber on either side of the IFC. The lid should remain on top of the hydration 25

30 Setting Up a Chip Run on the 4.96 Chip chamber throughout chip setup and chip scanning to maintain desired humidity. Controlled Free Interface Diffusion Protein and reagents each migrate from their respective chambers at a rate proportional to molecular weight. Reagents of low molecular weight such as salt ions, buffer, and polyethylene glycols (PEGs) migrate to the protein chamber faster than protein migrates to the reagent chamber. The controlled diffusion time assures that the optimum amount of reagent diffuses into the protein chamber with minimal diffusion of protein into the reagent chamber. Depending on the temperature used during diffusion and the OptiMix Reagents on the TOPAZ Chip, most reagents are fully equilibrated within min after diffusion begins, as shown in Table 6. Closing the interface valve at this time maintains a higher concentration of protein in the protein chamber. Initiating Diffusion At any temperature, after the T0 scan, run one of the controlled FID scripts shown in Table 5. Each controlled FID script initiates diffusion and then terminates FID after the appropriate controlled diffusion time has passed. Table 5 Equilibration Times for Controlled FID Temperature Equilibration Time (min) Controlled FID Script Room Temperature 60 3 FID Control RT 4 ºC FID Control 4C 26

31 Initiating Diffusion IMPORTANT Delivery of pressurized gas to the FID Crystallizer controller continues throughout the duration of any of the scripts listed in Table 5 on page 26. In laboratories that plumb compressed gas tanks to the FID Crystallizer controller, we recommend use of the 3 FID Start script in conjunction with the 4 FID End script to conserve compressed gas. As an alternative to the scripts shown in Table 5, you can run the 3 FID Start script to release pressure at the interface accumulator, open the interface valves and initiate diffusion. Use the 4 FID End script to stop FID after the recommended equilibration time. Use 3 FID Start and 3 FID End when you stagger the protocol steps, as shown in Figure 5 on page 13, to set up eight chips (3072 diffusion experiments) in approximately 4.5 hours. IMPORTANT If you run the 3 FID Start script, you must run the 4 FID End script to terminate diffusion after the appropriate equilibration interval. See Table 5 for the equilibration time recommended at each temperature. To initiate diffusion 1 In the FID Crystallizer software, enter the chip ID at a bay position. The chip ID is linked to Experiment and operator information that was previously entered. 2 Select either the appropriate controlled FID script, shown in Table 5, or the 3 FID Start script. 3 Place the 4.96 Chip on the corresponding FID Crystallizer controller bay, with the notched corner of the chip aligned with the A1 label in the bay. 4 Press Start. 5 When the script is completed, click Finish to gain access to the chip. At the end of a controlled FID script, diffusion has been initiated and, after appropriate diffusion time, FID has been terminated. 27

32 Setting Up a Chip Run on the 4.96 Chip Incubating Chips At the end of the 3 FID Start script, the interface valves are open and diffusion has been initiated. Run the 4 FID End script to terminate FID at the end of the recommended equilibration time, as shown in Table 5 on page When FID has been terminated, remove the TOPAZ Chip from the FID Crystallizer controller and incubate for crystal development. Incubation humidity and timing are dependent on the incubation temperature and OptiMix Reagent Kit used with the TOPAZ Chip. As shown in Table 6, the typical incubation time for a 4.96 Chip at room temperature is 7 days. Table 6 Controlled FID Times and Incubation Conditions for 4.96 Chips Room Temperature 4 ºC OptiMix Reagent Kit Controlled FID (min) %Humidity Incubation Time (days) Controlled FID (min) %Humidity Incubation Time (days) OptiMix OptiMix OptiMix OptiMix-PEG To maintain accumulator pressurization Repeat the 7 Recharge script every 7 days. IMPORTANT Recharge chip accumulators within 7 days of terminating FID. Inspecting for Crystals Protein crystallization is a process that relies on slow equilibration of the protein and the crystallization reagents. Monitor incubation on the TOPAZ Chip at regular intervals to identify crystal formation. We recommend performing a full scan of the 4.96 Chip at 24 hr., 48 hr., 4 days and 7 days after T0. 28

33 Inspecting for Crystals The TOPAZ AutoInspeX Workstation automates scanning and detection of crystals in all the chambers on a 4.96 Chip. If you do not have access to the TOPAZ AutoInspeX Workstation, you may use an inverted optical microscope to scan the diffusion chambers. 29

34 Setting Up a Chip Run on the 4.96 Chip 30

35 TROUBLESHOOTING Observation Possible Cause Recommended Action At the end of a script, the appropriate valves are not closed. Some chambers in the 4.96 Chip are not filled at the end of the 2 Load 4.96 script. At the pause at the end of reagent blindfill in a 5 Auto... script, some chambers in the 4.96 Chip are not filled. Obstacle prevents proper seal between chip and controller arm. Protein was not loaded before running the script. Reagent was not dispensed to one or more reagent inlets. Air bubbles in reagent channel or reagent chamber. Protein was not loaded before running the script. Reagent was not dispensed to one or more reagent inlets. Check that the chip is seated and flat on the controller bay, with the notch on the chip aligned to the A1 on the controller bay. Make sure the hydration chamber lid is flat and firmly seated on the chip. Check that the surface of the chip is flat and free of cracks. To dispense and load protein into chambers: a. Dispense at least 1.4 µl protein into the protein inlet, as described on page 22. b. Place the chip on the controller bay and run 6 Reload 4.96 script. To dispense and load reagent into chambers: a. Determine which reagent inlet was not filled. b. Dispense at least 10 µl reagent to the missed inlet, as described on page 19. c. Place the chip on the controller bay and run 6 Reload 4.96 script. Place the chip on the controller bay and run 6 Reload 4.96 script. To dispense and load protein into chambers: a. Dispense at least 1.4 µl protein into the protein inlet, as described on page 22. b. Extend the pause step until all chambers are filled. To dispense and load reagent into chambers: a. Determine which reagent inlet was not filled. b. Dispense at least 10 µl reagent to the missed inlet as described on page 19. c. Extend the pause step until all chambers are filled. Air bubbles in reagent channel. Extend the pause step until all chambers are filled. i

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37 ORDERING ADDITIONAL PARTS Contact Fluidigm Corporation at FLUIDLINE ( ) to order any of these additional items. Outside the U.S., call Fluidigm Part Number Item Description TPZ-M-4.96 TOPAZ 4.96 Chip (96 reagents, 4 proteins) Hydration Fluid Concentrate, 10 ml TPZ-R-OS1 OptiMix -1 Crystal Screening Reagents, 96 TPZ-R-OS2 OptiMix- 2 Crystal Screening Reagents, 96 TPZ-R-OS3 OptiMix -3 Crystal Screening Reagents, 96 TPZ-R-OSPE OptiMix- PEG Crystal Screening Reagents, TOPAZ FID Crystallizer User Guide Quick Reference Card, Features of 4.96 Chip TPZ-M-1.96 TOPAZ 1.96 Chip (96 reagents) iii

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39 INDEX INDEX Numerics 1 Prep script 19 2 Load 4.96 script Chip closing control valves 19 containment channel 3 control channels 3 diffusion experiment 4 diffusion, initiating 26 dispensing protein 23 dispensing reagents 19 figure 18 interface channel 3 interface valve 3 labels 18 numbering systems, Table 22 T0 24 A A1 at notched corner 22 C contamination prevention 8 controlled FID 26 O ordering parts iii P protein dispensing to 4.96 Chip 23 preparation and handling 8 pressurizing 4.96 Chip 24 preventing contamination 8 protein inlet 22 R reagent dispensing to 4.96 Chip 19 pressurizing 4.96 Chip 24 viscous 20 reagent inlet 19 S scripts, table of 14 staggering chip preparation 12 D diffusion experiment 6 E eight-channel pipette 20 H hydration chamber lid 17 I IFC 1 L lyophilization, and protein preparation 9 N notched corner 22 numbering systems on chip 7 v

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