Assay development guidelines and protocol notes

This topic explains each step of the protocol so that you can optimize the On‑Cartridge Reaction protocol to your particular experimental design.

For guidelines on performing a mock run, see About performing a mock run (optional).

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 10 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 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. Increase the number of wash cycles to clean the syringes better. However, more cycles increases the total run time, consumes more reagents, and causes wear on the syringes. Wash Cycles: • Default: 3 • Practical: 3–5 • Range: 1–10
Equilibrate	This step aspirates the Equilibration Buffer into the syringes and dispenses the buffer through the cartridges. The step ensures that the resin bed has been fully equilibrated. Ensure that you equilibrate the cartridge bed with a buffer that establishes optimal chemical conditions for the reaction during the Reaction step. In the case of an enzymatic reaction, the equilibration buffer would be the optimized enzyme reaction buffer. Ensure that the optimal reaction buffer does not break the affinity interaction holding the immobilized substrate on the resin bed. Always perform the Equilibrate step to establish optimum reaction conditions. Volume. The default volume, 50 µL, is equal to 10 column volumes, which should be sufficient for complete buffer exchange. • Default: 50 • Practical: 50–100 • Range: 0–250 Note: Setting the volume to zero will skip all tasks associated with the Equilibrate step except syringe washing at the wash station. Flow rate. A flow rate slower than the default rate, 10 µL/min, will likely have no benefit, but will increase the total assay time. A flow rate faster than 25 µL/min might not equilibrate through the pores in the beads across the full length of the cartridge bed. • Default: 10 • Practical: 5–25 • Range: 0.5–500 Wash cycles. 250 µL of DI water is used for each syringe wash cycle. • Default: 1 • Practical: 1–3 • Range: 0–10
Collect Flow Through	This step dispenses the flow-through during the Equilibrate step into Flow Through Collection plate (deck location 7) for downstream processing or for analysis. This step is used for optimizing or troubleshooting a protocol to ensure that the equilibration solution is not eluting the immobilized substrate off the cartridge. If the Collect Flow Through step is not selected, the flow-through from the Equilibrate step is dispensed directly into the wash station.
Reaction	This step aspirates the Reagent, from deck location 4, through the cartridges in two steps, and then aspirates a chase volume: 1 Initial Aspiration. Aspirates 4 µL of liquid at a flow rate of 10 µL/min (minimum volume). 2 Secondary Aspiration. Aspirates any additional volume at a flow rate appropriate to satisfy the Duration setting. For example, if the volume is set at 6 µL and the time was set for 30 minutes, the remaining 2 µL of the 6 µL volume would be aspirated at a flow rate of ~0.068 µL/min (2 µL / (30 min – 0.4 min)). 3 Reaction Chase Aspiration. Aspirates the chase at the specified volume and flow rate. The Reaction flow-through and Reaction Chase are collected in the syringes, and then dispensed into the Flow Through Collection plate (deck location 7), unless Combine with Eluate is selected. In which case, the Reaction flow-through and the Reaction Chase are dispensed into the Elution plate. Volume: The volume must be determined empirically. For an enzymatic reaction, the volume would depend on the enzyme. If the enzyme is robust enough to undergo the number of enzymatic cycles required to push the reaction to completion, 4 µL of enzyme may be sufficient. If the enzyme undergoes a limited number of cycles, the enzyme in the resin bed may need to be replenished during the course of the reaction, and significantly more than 4 µL might be required. • Default: 6 • Practical: 4–15 • Range: 4–100 Flow Rate: Initially, 4 µL is aspirated at 10 µL/min. Any additional volume is aspirated at a flow rate appropriate to satisfy the Duration setting. Wash Cycles: 250 µL of DI water is used for each syringe wash cycle. • Default: 1 • Practical: 1–3 • Range: 0–10
	Scenario using the default Reaction settings: 1 The Peltier Thermal Station heats until the set point temperature ±2°C is reached. 2 The Bravo 96AM Head picks up the cartridges from the Cartridge & Tip Seating Station (deck location 2). 3 The Reagent (deck location 4) is aspirated through the cartridges: 4 µL at a flow rate of 10 µL/min and 2 µL at a flow rate of 0.068 µL/min. (The Duration of both aspiration steps is 30 minutes.) 4 An external cartridge wash is conducted at the wash station. 5 25 µL Chase Buffer (deck location 3) is aspirated at 5 µL/min. 6 The cartridges are ejected into the Cartridge & Tip Seating Station. 7 The syringe contents are dispensed to the Flow Through Collection plate (deck location 7). 8 One internal syringe wash cycle is conducted.
Temperature	This setting specifies the set point temperature of the Peltier Thermal Station at deck location 4 during the Reaction step. The optimal temperature is a function of the reaction being conducted. The actual temperature in the cartridge will be less than this setting. Heat loss occurs as the heat transfers across the thermal plate insert, a small air gap between the plate insert and the wells of the plate, the plastic of the plate, the solution in the well, and the cartridge resin bed. For example, a temperature setting of 45 °C results in a cartridge bed resin temperature of approximately 37 °C using the Red PCR Plate Insert and a PCR plate. A thermal plate insert is critical when running a reaction at a temperature other than room temperature. Without the insert, the air gap between the Thermal Station and the wells of the plate results in a greater temperature differential between the setting in the application and the actual temperature in the cartridge bed. The temperature differential is not a constant percentage of the set point, so the preceding example should be considered only as a starting point for cartridge target temperatures other than 37 °C in the PCR plates. In addition, plate types vary in heat transfer efficiency because of their differences in design, such as well shape. IMPORTANT At least 3–5 µL of liquid is required in each well of a 96-well PCR plate at the end of the incubation time as this is the minimum volume required to maintain contact with the bottom of the cartridges. This contact is required to conduct heat into the resin bed. Longer incubation times or higher temperature settings require more volume, which should be determined empirically. A PCR plate is the most practical and common plate type at deck location 4 because of the low volumes of solution typically used at that location. The minimum volume required in the wells at the end of the incubation time for other plate types must be determined empirically. Example using Red PCR Plate Insert plus PRC plate. To conduct a 30-minute reaction at a resin bed temperature of approximately 37 °C with a 6 µL total aspiration volume, the temperature should be set to 45 °C and 12 µL of enzyme solution should be added per well before the run is started. 12 µL is required because 6 µL will be aspirated, 3 µL are expected to evaporate, and 3 µL are required at the end of the run to ensure heat conductance occurs during the entire run. Temperature °C: • Default: 25 • Practical: 20–60 • Range: 4–100
Duration	This setting specifies the total length of time to aspirate the Reagent (deck location 4) through the cartridges, including the initial and secondary aspiration steps. Note: This setting governs only the aspiration of Reagent through the cartridges. It does not include the time required to ramp up to temperature or the time required for the Reaction Chase step. Note: The duration of the reaction is related to the concentration of the reactant. In general, as the concentration of the reactant (for example, enzyme) decreases, the duration increases, as does the amount of reaction solution lost during the reaction due to evaporation, and the volume of excess reaction solution required. See the notes in the preceding Temperature step for more details. Time (minutes): • Default: 30 • Practical: 10–60 • Range: 1–180
Reaction Chase	If selected, this step aspirates the Chase Buffer (deck location 3) through the cartridges, to flush soluble reaction products into the syringe. This step occurs immediately after the aspiration of Reagent, combining the flow-through of the initial and secondary aspiration and the Chase Buffer within the syringes. Volume: This step is essentially an elution of the soluble reaction products. Elution of products off the AssayMAP cartridges is typically 2–3 column volumes (10–15 µL) when the affinity between eluate and the resin has been completely disrupted. Therefore, 10–15 µL should be a sufficient chase volume as long as there is no significant affinity between the soluble reaction product and the resin bed. The default volume is set to 5 column volumes to be conservative, but it is likely that you can decrease this volume. • Default: 25 • Practical: 10–30 • Range: 0–100 Note: Setting the volume to zero skips all Reaction tasks except for syringe washing. Flow Rate: • Default: 5 • Practical: 5–20 • Range: 0.5–500
Combine with Eluate	This setting specifies where the combination of the flow-through from the Reaction initial and secondary aspiration and the Chase Buffer will be collected: • If selected, collects the soluble reaction products at Eluate Collection (deck location 9). This is a good choice if the downstream analysis can easily separate the reactant, soluble, and eluted reaction products, allowing all the reaction products to be analyzed in a single run. • If not selected, collects the soluble reaction products at Flow Through Collection (deck location 7). This is a good choice if the goal is to analyze the soluble and immobilized reaction products separately because – Data on only one of these components is required, and there is not a good or rapid way of chromatographically separating the reactant, buffer, soluble, and immobilized reaction products in a single analytical run. – One wants to remove the buffer, reactant, and soluble reaction product from the immobilized reaction product, and elute this immobilized reaction product in a mass-spec-compatible solution. The setting is not selected by default.
Cup Wash 1	This step removes the residual liquid that may remain above the upper cartridge membrane after the Reaction step. The Cup Wash 1 step aspirates Cartridge Wash Buffer 1 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 removed at deck location 2 and then the liquid in the syringes is dispensed to the wash station. The syringes are washed at the wash station. Volume. A volume <10 µL might be insufficient for cup washing, while a volume >50 µL may offer little benefit. • Default: 25 • Practical: 25–50 • Range: 5–100 Wash cycle. Each cycle comprises one cup wash and one syringe wash. • Default: 2 • Practical: 1–3 • Range: 1–10 Note: Each wash cycle consists of a cup wash followed by syringe wash.
Internal Cartridge Wash 1	This step aspirates Cartridge Wash Buffer 1 from deck location 5 into the syringes, dispenses the buffer through the cartridges at the specified flow rate into the Flow Through Collection plate or wash station. The exterior of the cartridge tips are then washed at the wash station to remove any remaining samples. Volume. The volume to dispense through the cartridges to wash the resin bed. 10 column volumes (50 µL) is generally sufficient for an internal cartridge wash. Anything more than 20 column volumes (100 µL) has little benefit, increases the run length, and can result in lower recovery. • Default: 50 • Practical: 50–100 • Range: 0–250 Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except for syringe washing. Flow rate. A rate slower than the default flow rate, 10 µL/min, will have little benefit, but will increase the total assay time. A rate faster than 25 µL/min might not equilibrate through the pores in the beads across the full length of the cartridge bed, thus result in incomplete washing. • Default: 20 • Practical: 5–25 • Range: 0.5–500 Wash cycle. The number of syringe wash cycles to perform at the end of the task. 250 µL of DI water is used for each syringe wash cycle. • Default: 1 • Practical: 2–5 • Range: 0–10
Collect Flow Through (Internal Cartridge Wash 1)	This step dispenses the flow-through from Internal Cartridge Wash 1 directly into the Flow Through Collection plate (deck location 7). This step is typically for optimization or troubleshooting of a protocol to ensure that the wash solution is not eluting the immobilized substrate off the cartridge. If the Collect Flow Through step is not selected, the flow-through from Internal Cartridge Wash 1 is dispensed directly into the wash station.
Cup Wash 2	This step removes the residual liquid or buffer that may remain above the upper cartridge membrane after the Internal Cartridge Wash 1 step. The Cup Wash 2 step effectively rinses the cup portion of cartridges with buffer. This step aspirates Cartridge Wash Buffer 2 and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from samples or the previous cartridge wash is aspirated from the cartridge cups. Any cartridges that stuck to the probes during the cup wash are removed at deck location 2, and then the liquid in the syringes is dispensed to the wash station. The syringes are washed at the wash station. Volume. A volume <10 µL may be insufficient for cup washing, while a volume >50 µL may offer little benefit. • Default: 25 • Practical: 25–50 • Range: 5–100 Wash cycle. Each cycle comprises one cup wash and one syringe wash. • Default: 2 • Practical: 1–3 • Range: 1–10 Note: Each wash cycle consists of a cup wash followed by syringe wash.
Internal Cartridge Wash 2	This step aspirates Cartridge Wash Buffer 2 from deck location 6 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges at the specified flow rate into the Flow Through Collection plate or wash station. The exterior of the cartridge tips are washed at the wash station to remove any remaining sample, the cartridges are removed, and then the syringes are washed. Select the Internal Cartridge Wash 2 to provide an additional washing step to minimize non-target elements in the cartridge matrix, which could possibly contaminate the eluate. If used, this wash generally uses mass-spec-compatible solutions if the downstream analysis is mass spectrometry. Volume. The volume to dispense through cartridges to wash the resin bed. 10 column volumes (50 µL) is generally sufficient for an internal cartridge wash. Anything more than 20 column volumes (100 µL) has little benefit, increases the run length, and can result in lower recovery. • Default: 50 • Practical: 50–100 • Range: 0–250 Note: Setting the volume to 0 skips all Internal Cartridge Wash tasks except for syringe washing. Flow rate. A rate slower than the default flow rate, 10 µL/min, will have little benefit, but will increase the total assay time. A rate faster than 25 µL/min might not equilibrate through the pores in the beads across the full length of the cartridge bed, thus result in incomplete washing. • Default: 20 • Practical: 5–25 • Range: 0.5–500 Wash cycle. The number of syringe washes to perform at the end of the task after the cartridges are parked in the seating station. 250 µL of deionized water is used for each syringe wash cycle. • Default: 1 • Practical: 2–5 • Range: 0–10
Collect Flow Through (Internal Cartridge Wash 2)	This step dispenses the liquid eluted during Internal Cartridge Wash 2 directly into the Flow Through Collection plate (deck location 7). This step is typically only used for optimization or troubleshooting of a protocol to ensure that the wash solution is not eluting the immobilized substrate off the cartridge. If the Collect Flow Through step is not selected, the flow-through is dispensed directly into the wash station.
Stringent Syringe Wash	This step comprehensively cleans the syringes with the Elution Buffer prior to elution. The Stringent Syringe Wash step aspirates the Elution Buffer into the syringes, draws the buffer further into the syringe to ensure the entire syringe is rinsed, then dispenses the buffer into the wash station. The syringes are then washed at the wash station. Volume. The volume of Elution Buffer to aspirate and dispense. • Default: 50 • Practical: 50–100 • Range: 0–250 Note: Setting the volume to zero will skip all tasks associated with the Stringent Syringe Wash step. Wash cycle. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. • Default: 2 • Practical: 1–3 • Range: 1–10
Elute	This step uses Elution Buffer to elute immobilized reaction products from the cartridges. The Elute step aspirates the Elution Buffer into the syringes using the bare probes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Eluate Collection plate (deck location 9). An external cartridge tip wash is performed at the wash station to remove any sample on the outside of the cartridges, and then the cartridges are parked at the seating station. After the elution is complete, the syringes are used to mix the samples using 70% of the volume in the elution wells, if possible, or up to 250 µL per cycle in the Eluate Collection plate (deck location 9). The syringes are washed at the wash station for the specified number of wash cycles. Note: If the total volume in the Eluate Collection plate is <15 µL, the samples will not be mixed. You can also select the Eluate Discard and Existing Collection Volume, which are described in the following rows of this table. Volume. The maximum volume of eluate that can be collected in the Eluate Collection plate (deck location 9) is dependent on the maximum practical working volume of the labware at this location. The total volume in the Eluate Collection plate is determined by summing the net elution volume (Elute volume - Eluate Discard volume), the Reaction and Reaction chase volumes if Combine With Eluate Volume is selected, and the Existing Collection Volume. For labware-specific maximum volume, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. The elution volume required depends on the strength of the elution solution to break the affinity interaction between the immobilized substrate and the affinity ligand that is irreversibly linked to the resin bed. If the affinity interaction is completely broken by the elution solution, the immobilized reaction product can be eluted in 2–3 column volumes (10–15 µL). If the affinity interaction is not completely broken by the elution solution, the elution volume can increase but it is typically in the 20–30 µL range. Minimizing the elution volume can be very useful for downstream analysis, especially if the next steps require dilution or dry downs. • Default: 25 • Practical: 10–30 • Range: 0–250 Note: Setting the volume to zero will skip all tasks associated with the Elute step except syringe washing at the wash station. Flow rate. 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 elution 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: 0.1–500 Wash cycle. The number of syringe washes to perform at the wash station after an Elute step. 250 µL of DI water is used for each syringe wash cycle. • Default: 1 • Practical: 1–3 • Range: 0–10
Eluate Discard	This substep of the Elute step permits a specified volume of the first eluate from the cartridges to be discarded during the Elute step. The Elute step aspirates the Elution Buffer into the syringes (deck location 8), cartridges are mounted at the seating station (deck location 2), and Elution Buffer is dispensed through the cartridges at the specified Elute flow rate. The Eluate Discard volume is dispensed to the wash station (deck location 1). The remaining Elution Buffer is dispensed through cartridges at the Elute flow rate into the Eluate Collection plate (deck location 9). Example: If the Elute and Eluate Discard steps are selected with the following settings, Elute volume = 15 µL Eluate Discard volume = 2 µL the first 2 µL eluate from the cartridges will be discarded to the wash station, and the remaining 13 µL eluate will be collected in the Eluate Collection plate (deck location 9). Select the Eluate Discard step in a situation where minimizing the volume of eluate is critical. In the default protocol, this step is not selected. For some experimental situations, minimizing the total volume of sample eluates is critical to downstream processing. An example is when the concentration of organic solvent must be diluted to suitable levels for reversed-phase HPLC/MS analyses. In this case, it can be valuable to discard the first few microliters of eluate from an AssayMAP cartridge, which do not contain measurable amounts of analyte. For AssayMAP cartridges, the first 2 to 5 µL eluate contains little or no measurable amounts of analyte. The volume of the analyte-free eluate will vary depending on both the strength of the elution buffer and the degree of analyte saturation of the cartridges. This value should be determined empirically. Volume. The first volume of eluate that will be discarded during the Elute step. This value can equal, but cannot exceed the Elute volume. • Default: 0 • Practical: 2–5 • Range: 0–25 Flow rate. See notes for the Elute step.
Existing Collection Volume	This step enables you to specify an amount of liquid that is in each well of the Eluate Collection plate (deck location 9) at the start of the run. The Existing Collection Volume, the net volume from the Elute step (Elute volume - Eluate Discard volume), and the soluble reaction products, if Combine With Eluate is selected, feeds into logic that adjusts the well-bottom offset for sample elution, calculates the eluate mixing volume, and dynamically moves the head into and out of the wells during elution and eluate mixing in a volume-dependent manner. For the maximum practical working volumes of labware for eluate collection at deck location 9, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Select this option when the Eluate Collection plate contains a volume of liquid useful for immediately diluting the eluates, adjusting the pH of the eluates, or to aid in the recovery of small volumes of eluates from AssayMAP cartridges. Volume: • Default: 0 • Practical: 0–50 • Range: 0—300
Final Syringe Wash	Uses deionized water from the wash station to flush potential contaminants from the syringes. Each wash cycle aspirates 250 µL of fresh deionized water into the syringes and dispenses the fluid at an offset between the chimneys on the wash station. Increasing the number of Wash Cycles will clean the syringes better. However, increasing the value also increases the duration of the assay and affects wear on the syringes. Wash Cycles: • Default: 3 • Practical: 3–5 • 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 On-Cartridge Reaction protocol using the default protocol settings. Changing the selections or parameters will alter the movements.

 

Protocol step	Head moves to deck location...	Action
Initial Syringe Wash	2	Eject any cartridges off the head.
	1	Wash the syringes the specified number of cycles.
Equilibrate	3	Aspirate the Equilibration Buffer.
	2	Mount the cartridges on the head.
	1	Dispense the Equilibration Buffer through the cartridges to equilibrate. Wash the cartridge exteriors.
	2	Park the cartridges in the seating station.
	1	Wash the syringes at the wash station.
Reaction	4	Wait for Peltier Thermal Station to reach ±2 °C of the Reaction Temperature setting.
	2	Mount the cartridges on the head.
	4	Aspirate 4 µL at 10 µL/min.
	4	Aspirate the remaining volume (Reaction Volume setting in the form minus 4 µL) for the specified Duration (minutes) at the set Temperature (°C).
	1	Wash the cartridge exteriors at the wash station.
	3	Aspirate the specified volume of Chase Buffer at the specified Flow Rate (µL/min).
	2	Park the cartridges at the seating station.
	7	Dispense the flow-through into the Flow Through Collection plate. Mix the flow-through.
	1	Wash the syringes at the wash station.
Cup Wash 1	5	Aspirate cartridge Cartridge Wash Buffer 1 into the syringes.
	2	Perform Cup Wash.
	1	Dispense buffer at the wash station.
	1	Wash the syringes at the wash station for the specified number of cycles.
Internal Cartridge Wash 1	5	Aspirate Cartridge Wash Buffer 1 into the syringes.
	2	Mount the cartridges on the head.
	1	Dispense Cartridge Wash Buffer 1 through the cartridges for the Internal Cartridge Wash step.
	1	Wash the exterior of the cartridge tips at the wash station.
	2	Park the cartridges at the seating station.
	1	Wash the syringes at the wash station.
Stringent Syringe Wash	8	Aspirate the Syringe Wash Buffer (Elution Buffer).
	1	Dispense the buffer at the wash station.
	1	Wash the syringes at the wash station.
Elute	8	Aspirate the Elution Buffer.
	2	Mount the cartridges.
	9	Elute the samples into the Eluate Collection plate.
	1	Wash the cartridge exteriors at the wash station.
	2	Park the cartridges at the seating station.
	9	Mix eluates.
	1	Wash the syringes at the wash station.
Final Syringe Wash	1	Wash the syringes at the wash station.

 

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