Assay development guidelines and protocol notes
This topic explains the following:
• Each step of the protocol so that you can optimize the IMAC Cartridge Customization protocol to your particular experimental design
• Automation movements during the protocol
For details on how to use the Experiments Editor, see Using Experiment IDs.
Protocol stepwise guidelines
Protocol step | Guidelines and notes |
|---|---|
Number of Full Columns of Cartridges | This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct. If the column selection is: • Greater than the actual number of columns used, the Bravo Platform will apply too much force when mounting the cartridges, which can damage both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required. • Less than the actual number of columns used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable due to liquid moving past the syringe cartridges seal into the cartridge cup. Default: 1 Range: 1-12 |
Initial Syringe Wash | This step flushes any potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the head aspirates 250 µL into the syringes from the wash station chimneys and then moves by a fixed offset between the chimneys to dispense to waste. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. • Default: 3 • Practical: 3–5 • Range: 0–10 |
Prime | This step removes entrained air from the resin bed and properly wets the surface of the resin. In preparation for priming, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the Metal Waste plate, 10 µL of Priming Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Prime step aspirates the Priming Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Metal Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station. For the most effective priming of AssayMAP Fe(III)-NTA cartridges, the Priming Buffer requires that the solution contain at least 50% organic solvent. Higher concentrations of organic solvent are also acceptable. This step is selected by default. Volume (µL). The default volume is sufficient to wet and remove entrained air from the resin bed. Using less than the default volume may leave air in the resin bed. Using more than the default volume is unnecessary and increases run time. • Volume for 5 µL cartridge: – Default: 100 – Practical: 100–250 – Range: 0–250 • Volume for 25 µL cartridge: – Default: 250 – Practical: 250 – Range: 0–250 Note: Setting the volume to zero skips all Prime tasks except syringe washing. Flow rate (µL/min). A flow rate slower than the default value diminishes the ability to effectively remove entrained air from the cartridge. A flow rate faster than the default is not required and has not been tested. • Default: 300 • Practical: 300 • Range: 0.5–500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 µL of DI water is used for each syringe wash cycle. • Default: 1 • Practical: 1–3 • Range: 0–10 |
Strip Metal | This step removes the metal coordinated to the Fe(III)-NTA resin by using a strong chelator, such as EDTA. Complete removal of the bound metal from the resin bed is critical so that a different metal can be bound at the maximum metal-binding capacity of the cartridge. In preparation for stripping, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the Metal Waste plate, 10 µL of Priming Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Strip Metal step aspirates the specified volume of Metal Stripping Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through cartridges into the Metal Waste plate or into the Flow Through Collection plate. The exterior of the cartridge tips are washed at the wash station to remove any residual buffer on the exterior of the cartridges, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is selected by default. Volume (µL). The default volume is equal to 10 column volumes, which should be sufficient for complete removal of bound metal using a sufficiently concentrated chelator in the Metal Stripping Buffer. The default volume is probably much higher than required to strip the metal from the cartridges, but lower volumes have not been tested. • Volume for 5 µL cartridges: – Default: 50 – Practical: 50–100 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 250 – Practical: 250 – Range: 0–250 Note: Setting the volume to zero skips all Strip Metal step tasks except syringe washing. Flow rate (µL/min). A flow rate slower than the default will likely have no benefit, but will increase the total assay time. A flow rate > 15 µL/min using the default volume might not permit full penetration into the pores of the resin across the full length of the cartridge bed leaving residual metal on the cartridge. • Default: 5 • Practical: 2–15 • Range: 0.5–500 Wash cycle.The number of syringe wash cycles to perform at the end of this step. 250 µL of DI water is used for each syringe wash cycle. • Default: 3 • Practical: 2–5 • Range: 0–10 |
Collect Flow Through | If this step is selected, the flow-through from the Strip Metal step is dispensed directly into the Flow Through Collection plate. If this step is not selected, the flow-through is dispensed directly into the Metal Waste plate. This step is not selected by default. |
Cup Wash 1 | This step removes the small volume of residual liquid that might remain above the resin bed after the Strip Metal step. The Cup Wash step aspirates Cartridge Wash Buffer 1 into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the Metal Waste plate, and then washing the syringes at the wash station. This step is selected by default. Volume (µL). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 µL may offer little benefit. • Default: 25 • Practical: 25–50 • Range: 0–100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. • Default: 3 • Practical: 3–5 • Range: 0–10 |
Internal Cartridge Wash 1 | This step uses Cartridge Wash Buffer 1 to wash any remaining Metal Stripping Buffer from the resin bed and equilibrate the resin bed with a buffer compatible with the Metal Reagent to be used in the Load Metal step. In preparation for Internal Cartridge Wash 1, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the Metal Waste plate, 10 µL of Cartridge Wash Buffer 1 is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer 1 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or the Metal Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is selected by default. Volume (µL). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. • Volume for 5 µL cartridges: – Default: 50 – Practical: 50–100 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 250 – Practical: 250 – Range: 0–250 Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (µL/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 µL/min may not equilibrate through the pores in the beads, resulting in incomplete washing. • Default: 10 • Practical: 5–20 • Range: 0.5–500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 µL of DI water is used for each syringe wash cycle. • Default: 3 • Practical: 2–5 • Range: 0–10 |
Collect Flow Through | If this step is selected, the flow-through from the Internal Cartridge Wash 1 step is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from Internal Cartridge Wash 1 is dispensed into the Metal Waste plate. This step is not selected by default. For the maximum practical working volumes per well for specific labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. |
Load Metal Reagent | This step allows the target metal to bind to the surface chemistry of the resin bed. No liquid is removed or added to the cartridge cups before the metal loading begins. The assumption is that there is still liquid in the cups from the wash step that will prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. This step aspirates Metal Reagent into the syringes, and then performs an external syringe wash at the wash station to remove any Metal Reagent remaining on the outside of the probes before mounting the cartridges. The Metal Reagent is dispensed through the cartridges into the Flow Through Collection plate or the Metal Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any Metal Reagent on the exterior of the cartridges, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is selected by default. Volume (µL). The volume of the Metal Reagent should be balanced with the concentration of the Metal Reagent and the metal binding capacity of the cartridge to ensure a large molar excess of metal to metal binding sites in the resin bed. • Default: 100 • Practical: 50–150 • Range: 0–250 Note: Setting the volume to zero skips all Load Metal Reagent tasks except syringe washing. Flow rate (µL/min). A flow rate less than the default will likely have no benefit, but will increase the total assay time. A flow rate > 15 µL/min using the default volume might not permit full penetration into the pores of the resin across the full length of the resin bed, leaving portions of uncharged NTA in the resin bed, which would reduce binding capacity. • Default: 5 • Practical: – 2–10 (5 µL cartridges) – 5–20 (25 µL cartridges) • Range: 0.1–500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 µL of DI water is used for each syringe wash cycle. • Default: 3 • Practical: 2–5 • Range: 0–10 |
Collect Flow Through | If this step is selected, the flow-through from the Load Metal Reagent step is dispensed into the Flow Through Collection plate. If this step is not selected, the flow-through from the Load Metal Reagent step is dispensed into the Metal Waste plate. This step is not selected by default. |
Cup Wash 2 | This step removes the residual solution that may remain above the resin bed after the Load Metal Reagent step. The Cup Wash 2 step aspirates Cartridge Wash Buffer 2 into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the Metal Waste plate, and then washing the syringes at the wash station. This step is selected by default. Volume (µL). A volume less than the default might be insufficient for cup washing, while a volume >50 µL may offer little benefit. • Default: 25 • Practical: 25–50 • Range: 0–100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. • Default: 3 • Practical: 3–5 • Range: 0–10 |
Internal Cartridge Wash 2 | This step uses Cartridge Wash Buffer 2 to wash unbound metal from the resin bed. In preparation for Internal Cartridge Wash 2, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the Metal Waste plate, 10 µL of Cartridge Wash Buffer 2 is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer 2 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or the Metal Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is selected by default. Volume (µL). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. • Volume for 5 µL cartridges: – Default: 50 – Practical: 50–100 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 250 – Practical: 250 – Range: 0–250 Note: Setting the volume to 0 skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (µL/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 µL/min might not equilibrate through the pores in the beads, resulting in incomplete washing. • Default: 10 • Practical: 5–20 • Range: 0.5–500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 µL of DI water is used for each syringe wash cycle. • Default: 3 • Practical: 2–5 • Range: 0–10 |
Collect Flow Through | If this step is selected, the flow-through from the Internal Cartridge Wash 2 step is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from the Internal Cartridge Wash 2 step is dispensed directly into the Metal Waste plate. This step is not selected by default. |
Final Syringe Wash | This step uses the wash station to flush potential contaminants from the syringes. Before the Final Syringe Wash begins, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the Metal Waste plate. No solution is added into the cartridge cups. Note: If the Final Syringe Wash is skipped, 10 µL of Cartridge Wash 2 will remain in the cartridge cups. During each Final Syringe Wash cycle, the head aspirates 250 µL of DI water into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. In cases where carryover is a major concern, increasing the number of wash cycles may provide marginally improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: • Default: 3 • Practical: 3–5 • Range: 0–10 |
Automation movements during the protocol
This section describes the basic automation movements of the AssayMAP Bravo Platform during the protocol using the default method. Changing the selections or parameters will alter the movements.
Protocol step | Head moves to deck location... | Action |
|---|---|---|
Starting protocol | 2 | Parks all cartridges that might have been loaded on the head from a protocol that had been previously aborted. |
1 | Dispenses any liquid remaining in the syringes into the wash station. | |
Initial Syringe Wash | 1 | Washes the syringes for 3 cycles. |
Prime | 2 | Aspirates 20 µL of air above this location, moves down to just above the cartridge engagement point and aspirates 60 µL, and then exercises the cartridges off task. |
3 | Dispenses into the Metal Waste plate. | |
1 | Washes the exterior of the syringe probes. | |
4 | Aspirates 10 µL of Priming Buffer for the cartridge air-gap-prevention step. | |
2 | Dispenses the 10 µL of buffer into the cartridge cups and exercises the cartridges off task. | |
4 | Aspirates the Priming Buffer. | |
2 | Mounts the cartridges onto the head. | |
3 | Dispenses the Priming Buffer through the cartridges into the Metal Waste plate to prime the cartridges. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
Strip Metal | 5 | Aspirates the Metal Stripping Buffer. |
2 | Mounts the cartridges on the head. | |
3 | Dispenses the Metal Stripping Buffer through the cartridges into the Metal Waste plate. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
Cup Wash 1 | 6 | Aspirates the Cartridge Wash Buffer 1 into the syringes. |
2 | Washes the cartridge cups and exercises the cartridges off task. | |
3 | Dispenses the Cartridge Wash buffer 1 into the Metal Waste plate. | |
1 | Washes the syringes. | |
Internal Cartridge Wash 1 | 2 | Aspirates 20 µL of air above this location, moves down to just above the cartridge engagement point and aspirates 60 µL, and then exercises the cartridges off task. |
3 | Dispenses into the Metal Waste plate. | |
1 | Washes the exterior of the syringe probes. | |
6 | Aspirates 10 µL of Cartridge Wash Buffer 1 for the cartridge air-gap-prevention step. | |
2 | Dispenses the 10 µL of buffer into the cartridge cups and exercises the cartridges off task. | |
6 | Aspirates the Cartridge Wash Buffer 1 into the syringes. | |
2 | Mounts the cartridges on the head. | |
3 | Dispenses the Cartridge Wash Buffer 1 through the cartridges into the Metal Waste plate. | |
1 | Washes the exterior of the cartridge tips at the wash station. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
Load Metal Reagent | 9 | Aspirates the Metal Reagent into the syringes. |
1 | Washes the exterior of the syringe probes. | |
2 | Mounts the cartridges on the head. | |
3 | Dispenses the Metal Reagent through the cartridges into the Metal Waste plate. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
Cup Wash 2 | 8 | Aspirates the Cartridge Wash Buffer 2 into the syringes. |
2 | Washes the cartridge cups and exercises the cartridges off task. | |
3 | Dispenses the Cartridge Wash buffer 2 into the Metal Waste plate. | |
1 | Washes the syringes. | |
Internal Cartridge Wash 2 | 2 | Aspirates 20 µL of air above this location, moves down to just above the cartridge engagement point and aspirates 60 µL, and then exercises the cartridges off task. |
3 | Dispenses into the Metal Waste plate. | |
1 | Washes the exterior of the syringe probes. | |
8 | Aspirates 10 µL of Cartridge Wash Buffer 2 for the cartridge air-gap-prevention step. | |
2 | Dispenses the 10 µL of buffer into the cartridge cups and exercises the cartridges off task. | |
8 | Aspirates Cartridge Wash Buffer 2 into the syringes. | |
2 | Mounts the cartridges on the head. | |
3 | Dispenses Cartridge Wash Buffer 2 through the cartridges into the Metal Waste plate. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
Final Syringe Wash | 2 | Moves down to just above the cartridge engagement point and aspirates 60 µL, and then exercises the cartridges off task. |
3 | Dispenses into the Metal Waste plate. | |
1 | Washes the syringes. |