Assay development guidelines and protocol notes

This topic explains each step of the Peptide Cleanup: Aspiration Mode protocol so that you can optimize the protocol to your particular experimental design.

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 removes potential contaminants from the syringes. 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 the syringe contents to waste. Increase the number of wash cycles to better clean the syringes. However, be aware that increasing the number of wash cycles also increases protocol run time and reduced syringe lifespan. This step is selected by default. Wash Cycles: • Default: 5 • Practical: 3–5 • Range: 1–10
Prime	This step removes entrained air from within the cartridges and properly wets the surface of the resin. Priming of AssayMAP reversed-phase cartridges requires a buffer that contains at least 25% organic solvent. Higher concentrations of organic solvent are also acceptable. The Prime step aspirates the 100 µL Priming and Syringe Wash Buffer into the syringes, mounts the cartridges on the syringes, and then dispenses the buffer through the cartridges at 300 µL/min into the Organic Waste plate. After parking the cartridges at the tip seating station, the syringes are washed at the wash station using 250 µL of water per wash cycle.
Equilibrate	Ensure that you equilibrate the cartridge bed with a buffer that establishes optimal chemical conditions for binding during the Load Samples step. This step aspirates the Equilibration Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges at the specified flow rate into the wash station. After parking the cartridges in the tip seating station, the syringes are washed at the wash station using 250 µL of water. This step is selected by default. Volume (µL). The default volume is equal to 10-column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. • Default: 50 • Practical: 50–100 • Range: 0–250 Note: Setting Volume at 0 skips all Equilibrate tasks except for syringe washing. Flow Rate (µL/min). A flow rate slower than 10 µL/min will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 µL/min might not equilibrate through the pores in the beads across the full length of the cartridge bed. • Default: 25 • Practical: 5–25 • Range: 0.5–500 Wash Cycles: • Default: 0 • Practical: 1–3 • Range: 0–10
Load Samples	This step mounts the cartridges on the syringes, aspirates the samples through the cartridges, and performs an external cartridge tip wash at the wash station to remove any sample on the outside of the cartridge. This wash is followed by aspirating 5 µL of Equilibration Buffer (sample chase) through the cartridges at the Load Samples flow rate. The sample chase ensures that the sample volume in the cartridges at the end of the sample load moves through the cartridge bed at the same rate as the rest of the sample. Finally, the cartridges are parked at the seating station and the flow-through plus sample chase are collected in the Flow Through Collection plate. The protocol accommodates sample volumes up to 245 μL. Although, the form permits you to enter smaller volumes, the minimum advisable sample volume to be loaded onto an AssayMAP cartridge is 10 μL. IMPORTANT The volume of sample to load should be balanced with the sample concentration and the mass capacity of the cartridge. Be sure to include the recommended labware-dependent volume overage to prevent air from entering the cartridge. For more information, see Transferring the samples to the microplate. This step is selected by default. Volume (µL). The volume of sample should be balanced with the sample concentration and the mass capacity of the cartridge. To determine the volume of sample to load, see Determining the volume of sample to load. • Default: 50 • Practical: 25–245 • Range: 0–245 Note: Setting Volume at 0 skips all Load Samples tasks except for syringe washing. Flow rate (µL/min). The optimum sample loading flow rate requires balancing the speed of the assay and desired recovery. When setting the flow rate, be aware that the quantitative binding capacity is inversely proportional to the flow rate. For some samples, a flow rate greater than 15 µL/min can result in a low dynamic binding capacity. It might also produce some loss of total binding capacity, as the sample does not have sufficient time to diffuse throughout the pores of the cartridge resin bed. Using flow rates below 5 µL/min might not significantly increase analyte binding. • Default: 5 • Practical: 2–15 • Range: 0.5–500 Wash Cycles. 250 µL of DI water is used for each syringe wash cycle. • Default: 5 • Practical: 2–5 • Range: 0–10
Cup Wash	This step removes the residual sample liquid that may remain above the upper cartridge membrane after the Load Samples step. The Cup Wash step aspirates Cartridge Wash Buffer and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from samples is aspirated from the cartridge cups. Any cartridges that stuck to the probes during the cup wash are parked in the seating station and then the liquid in the syringes is dispensed into the wash station. The final operation is a syringe wash at the wash station. This step is selected by default. Volume (µL). A volume <10 µL might be insufficient for cup washing, while a volume >50 µL may offer little benefit. • Default: 50 • Practical: 25–50 • Range: 1–100 Wash Cycles: • Default: 2 • Practical: 1–3 • Range: 1–10 Note: Each wash cycle consists of a cup wash followed by syringe wash.
Internal Cartridge Wash	This step uses Cartridge Wash Buffer to wash non-specifically bound molecules from the cartridge. The Internal Cartridge Wash step mounts the cartridges, aspirates Cartridge Wash Buffer through the cartridges, parks the cartridges, dispenses the contents of the syringes into the wash station at an offset from the chimneys, and then washes the syringes using 250 µL of DI water for each syringe wash cycle. Volume (µL). The volume to dispense through the cartridges to wash the resin bed. Volumes higher than the default volume may improve the purification marginally but also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. • Default: 50 • Practical: 50–100 • Range: 0–250 Note: Setting Volume at 0 skips all Internal Cartridge Wash tasks except for syringe washing. Flow Rate (µL/min). A rate slower than 10 μL/min will likely have no 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: 25 • Practical: 5–25 • Range: 0.5–500 Wash Cycles. The number of syringe washes to perform at the end of the task. 250 µL of DI water is used for each syringe wash cycle. • Default: 3 • Practical: 2–5 • Range: 0–10
Stringent Syringe Wash	This step cleans the syringes with Priming & Syringe Wash Buffer prior to elution. The Stringent Syringe Wash step aspirates the buffer into the syringes and then dispenses the buffer into the Organic Waste plate. The syringes are then washed at the wash station. This step is selected by default. Volume (µL): The volume of Priming & Stringent Syringe Wash Buffer to aspirate and dispense. Volumes lower than the default volume may be insufficient for efficient syringe washing. • Default: 50 • Practical: 50–100 • Range: 1–250 Note: Setting Volume at 0 skips the Stringent Syringe Wash step. Wash Cycles. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. • Default: 1 • Practical: 1–3 • Range: 1–10
Elute	This step uses Elution Buffer to elute bound analytes from the cartridges. The Elute step aspirates the Elution Buffer into the syringes, washes the probes at the wash station, and then mounts the cartridges and dispenses the buffer through the cartridges at the specified flow rate into the Eluate Collection plate. The exterior of the cartridge tips are then washed at the wash station before parking the cartridges at the tip seating station. Using the syringes, the samples are mixed in the Eluate Collection plate. The syringes are washed at the wash station for the specified number of wash cycles, using 250 µL per cycle. Note: If the total volume in the Eluate Collection plate is <15 µL, the samples will not be mixed. This step is selected by default. Volume (µL). Using sufficiently strong elution conditions and the default flow rate (5 µL/min), peptides can be fully eluted from cartridges in approximately two cartridge column volumes. • Default: 25 • Practical: 10–30 • Range: 0–250 Note: Setting Volume at 0 skips all Elute tasks except for syringe washing. Flow Rate (µL/min). A flow rate slower than 5 µL/min is unlikely to improve the elution yield. Elution yield may be compromised if flow rates are faster than 15 µL/min for a given volume of elution buffer (that is, more buffer may be required to get the same elution yield at high elution flow rates relative to using lower flow rates for a given elution volume). • Default: 5 • Practical: 2–15 • Range: 0.5–500 Wash Cycles. The number of syringe washes to perform at the wash station after an Elute step. 250 ?L of DI water is used for each syringe wash cycle. • Default: 0 • Practical: 1–3 • Range: 0–10
Re-Equilibrate	This step uses Equilibration Buffer to return the cartridge to a low percentage organic solvent condition. The step aspirates the buffer into the syringes, mounts the cartridges, dispenses the buffer through the cartridges into the wash station, and then parks the cartridges. Volume (µL). The default volume is equal to 10-column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. • Default: 50 • Practical: 50–100 • Range: 0–250 Flow Rate (µL/min). A flow rate slower than 10 µL/min will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 µL/min using the default volume might not equilibrate through the pores in the beads. • Default: 25 • Practical: 5–25 • Range: 0.5–500
Final Syringe Wash	This step uses the wash station to flush potential contaminants from the syringes. During each Final 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 the syringe contents to waste. Note: The amount of carryover in the syringe (expressed as dilution of the starting sample) depends on the total number of syringe wash cycles. In cases where carryover is a major concern, increasing the number of wash cycles also increases the dilution factor, but with a cost of increased assay time and reduced syringe lifespan. This step is selected by default. Wash Cycles: • Default: 3 • Practical: 1–3 • Range: 1–10

 

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Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Peptide Cleanup: Aspiration Mode protocol using the default settings. Changing the selections or parameters will alter the movements.

 

Protocol step	Head moves to deck location...	Action
Start protocol	2	Checks the head and park all cartridges that might have been mounted on the syringes 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 the specified number of cycles.
Prime	5	Aspirates the Priming and Syringe Wash Buffer for priming.
	2	Mounts the cartridges on the syringe probes.
	3	Dispenses the buffer through the cartridges and into the Organic Waste plate to prime the cartridges.
	2	Parks the cartridges.
	1	Washes the syringes.
Equilibrate	8	Aspirates the Equilibration and Cartridge Wash Buffer.
	2	Mounts the cartridges on the syringe probes.
	1	Dispenses the buffer through the cartridges to waste at the wash station.
	1	Washes the exterior of the cartridge tips at the wash station.
	2	Parks the cartridges.
	1	Washes the syringes.
Load Samples	2	Mounts the cartridges on the syringe probes.
	4	Aspirates the samples through the cartridges into the syringes.
	1	Washes the exterior of the cartridge tips.
	8	Aspirates 5 µ of Equilibration and Cartridge Wash Buffer for the sample chase.
	2	Parks the cartridges.
	7	Dispenses the flow-through plus sample chase into the Flow Through Collection plate.
	1	Washes the syringes.
Cup Wash	8	Aspirates the Equilibration and Cartridge Wash Buffer.
	2	Washes the cartridge cups.
	1	Dispenses the Cartridge Wash Buffer.
	1	Washes the syringes.
Internal Cartridge Wash	2	Mounts the cartridges on the syringe probes.
	8	Aspirates the Equilibration and Cartridge Wash Buffer into the syringes.
	1	Dispenses the buffer to waste at the wash station.
	2	Parks the cartridges.
	1	Washes the syringes at the wash station.
Stringent Syringe Wash	5	Aspirates the Syringe Wash Buffer.
	3	Dispenses the syringe contents into the Organic Waste plate.
	1	Washes the syringes at the wash station.
Elute	6	Aspirates the Elution Buffer into the syringes.
	1	Washes the syringe probes at the wash station.
	2	Mounts the cartridges on the syringe probes.
	9	Elutes the samples into the Eluate Collection plate.
	1	Washes the cartridge exteriors.
	2	Parks the cartridges at the tip seating station.
	1	Washes the syringes at the wash station.
	9	Uses the syringes to mix the samples in the Eluate Collection plate.
	1	Washes the syringes at the wash station.
Re-Equilibrate	8	Aspirates Equilibration Buffer into the syringes.
	2	Mounts the cartridges on the syringe probes.
	1	Dispenses the syringe contents through the cartridges to waste at the wash station.
	1	Washes the exterior cartridge tips at the wash station.
	2	Parks the cartridges in the seating station.
Final Syringe Wash	1	Washes the syringes at the wash station.

 

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