Assay development guidelines and protocol notes

This topic explain each step of the protocol so that you can optimize the Immobilization 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 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. Increase the number of wash cycles to clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. Wash Cycles: • Default: 3 • Practical: 3–5 • Range: 0–10
Prime	This step removes entrained air from within the cartridges and properly wets the surface of the resin. The AssayMAP affinity purification cartridges (PA-W, SA‑W, and PG‑W) typically used with this application contain proteins covalently immobilized onto resin. These cartridge types should be primed with aqueous buffers containing low or no amounts of organic solvent or protein denaturants. Because the Equilibration Buffer is drawn from the same reservoir as the Priming Buffer, buffers that favor analyte binding should be used for both priming and equilibration. Before the priming begins, 10 µL of Priming Buffer is aspirated and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. Volume (µL). The default volume should be sufficient to wet the cartridge resin and remove entrained air from within the cartridge. 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 cartridges: – Default: 100 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 250 – Range: 0–250 Note: Setting the volume to zero will skip all tasks associated with the Prime step except syringe washing at the wash station. 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: 250–350 • 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
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 by using a slower flow rate than the priming step. Ensure that you equilibrate the cartridge bed with a buffer that establishes optimal chemical conditions for binding during the Load Samples step. Always perform the Equilibrate step after cartridge priming to establish optimum sample loading conditions. 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. • Volume for 5 µL cartridges: – Default: 50 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 250 – Range: 0–250 Note: Setting the volume to zero will skip all Equilibrate step tasks except syringe washing. Flow rate (µL/min). A flow rate slower than the default rate 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. • Default: 10 • Practical: 5–20 • 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
Load Samples	This step aspirates the sample into the syringes, washes the syringe exterior, mounts the cartridges, and then dispenses the samples through the mounted 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 samples, the cartridges are parked, and the syringes are washed at the wash station. The protocol accommodates sample volumes up to 1000 µL to be dispensed through the cartridges. Although, the form permits you to enter smaller volumes, the minimum advisable sample volume to be loaded onto an AssayMAP cartridge is 10 µL. Each syringe has a maximum capacity of 250 µL. When sample volumes are greater than 250 µL, the protocol will iteratively load samples onto cartridges in equal volume steps. To determine the number and volume of the iterative load steps, the protocol uses the following formulas: • # of times to load = total sample volume/250, where # times to load is rounded up to nearest integer • volume of each load = sample volume/# of times to load For example, if the total sample volume is 900 µL, then: # times to load = 900/250 = 3.6, which is rounded up to 4 volume of each load = 900/4 = 225 If Collect Flow Through is selected for the Load Samples step, be sure that the Flow Through Collection plate has sufficient maximum well capacity. For details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. 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 Preparing the sample plates. To determine the volume of sample to load, see Determining the volume of sample to load. This step is selected by default.
	Volume (µL): • Default: 100 • Range: 0–1000 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 faster than 15 µL/min can result in a loss of total binding capacity in addition to a loss of quantitative capacity, as the sample does not have sufficient time to diffuse throughout the pores of the cartridge resin bed. Using flow rates slower than 5 µL/min might not significantly increase analyte binding, but this is highly dependent on the molar ratio of the capture ligand compared to the target molecule. • Default: 5 • Practical: 2–15 • Range: 0.1–500 Wash cycles. 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 sample flow-through from the Load Samples step is dispensed in the Flow Through Collection plate at deck location 7. If the Collect Flow Through step is not selected, the flow-through from the Load Samples step is dispensed directly into the wash station. The Collect Flow Through step is skipped if the Load Samples step is not conducted. This step is selected by default.
Cup Wash 1	This step removes the residual sample liquid that may remain above the upper cartridge membrane after the Load Samples 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 the seating station and then the liquid in the syringes is dispensed to the wash station. The syringes are washed at the wash station. Volume (µL). 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: 0–100 Note: Setting the volume to zero will skip all tasks associated with the Cup Wash step. Wash cycle. Each cycle comprises one cup wash and one syringe wash. • Default: 3 • 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 uses Cartridge Wash Buffer 1 to wash non-specifically bound molecules from the cartridge resin bed. Before the cartridge wash begins, 10 µL of Cartridge Wash Buffer 1 is apirated 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, 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. If the Load Samples step was selected, 25 µL of Cartridge Wash Buffer 1 is aspirated in addition to the specified volume for the Internal Cartridge Wash 1. This additional volume is the sample chase and it is dispensed 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. If the wash volume plus the sample chase equal >250 µL, the sample chase is performed with part of the wash volume. This step is selected by default. 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. • Volume for 5 µL cartridges: – Default: 50 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 250 – 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 the default flow rate, 10 µL/min, will 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 the task. 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 Internal Cartridge Wash 1 is dispensed in the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from Internal Cartridge Wash 1 is dispensed at the wash station. This step is not selected by default.
Load Blocking Reagent	This step allows a reagent of defined composition to be flowed through the cartridge after ligand immobilization to help minimize non-specific binding in subsequent target purification steps. The Load Blocking Reagent step may be especially helpful when a sub-saturating amount of ligand is bound to the resin in the cartridge. The Load Blocking Reagent step aspirates Blocking Reagent into the syringes, performs an external syringe wash at the wash station, mounts the cartridges, dispenses the Blocking Reagent through the cartridges at the specified flow rate to either the Flow Through Collection plate or the wash station. An external cartridge tip wash is performed at the wash station where cartridge tips dip into the wash station chimneys to remove any blocking reagent on the outside of the cartridge. The cartridges are parked at the seating station, and then the syringes are washed with DI water for the specified number of wash cycles. Select the Load Blocking Reagent step to minimize to non-specific binding during subsequent target purification steps. This step is selected by default. Volume (µL). The volume of Blocking Reagent to aspirate into the syringes and dispense through the cartridges. • Default: 100 • Range: 0–250 Note: Setting the volume to zero will skip all tasks associated with the Load Blocking Reagent step except syringe washing at the wash station. Flow rate (µL/min). A rate slower than 5 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, which may result in incomplete blocking. • Default: 5 • Practical: 5–15 • Range: 0.1–500 Wash cycles. 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 liquid eluted during the Load Blocking Reagent step is dispensed in the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from the Load Blocking Reagent step is dispensed at the wash station. Select this step if you want to collect the Blocking Reagent flow-through. This step is not selected by default.
Cup Wash 2	This step removes the residual buffer that may remain above the upper cartridge membrane after the Load Blocking Reagent 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 previous steps is aspirated from the cartridge cups. Any cartridges that stuck to the probes during the cup wash are removed at the seating station, and then the liquid in the syringes is dispensed to the wash station. The syringes are washed at the wash station. Volume (µL). 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: 0–100 Note: Setting the volume to zero will skip all tasks associated with the Cup Wash 2 step. Wash cycle. Each cycle comprises one cup wash and one syringe wash. • Default: 3 • 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 uses Cartridge Wash Buffer 2 to wash non-specifically bound molecules from the cartridge resin. Before the cartridge wash begins, 10 µL of Cartridge Wash Buffer 2 is aspirated into the syringes and 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 at the specified flow rate into the Flow Through Collection plate or wash station. An external cartridge tip wash is performed at the wash station where cartridge tips dip into the wash station chimneys to remove any sample on the outside of the cartridge. The cartridges are parked at the seating station, and then the syringes are washed with DI water for the specified number of wash cycles. Select the Internal Cartridge Wash 2 to provide an additional washing step to minimize non-target elements from the cartridge, which could possibly contaminate the eluate. This step is selected by default. Volume (µL). The volume to dispense through cartridges to wash the resin bed. • Volume for 5 µL cartridges: – Default: 50 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 250 – 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 the default flow rate, 10 µL/min, will 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 washes to perform at the end of the task after the cartridges are parked in the seating station. 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 liquid eluted during Internal Cartridge Wash 2 is dispensed in the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through is dispensed at the wash station. Select this step if you want to collect the flow-through generated during Internal Cartridge Wash 2. This step is not selected by default.
Stringent Syringe Wash	This step cleans the syringes with the Stringent Syringe Wash Buffer. This step aspirates the buffer into the syringes, draws the buffer further into the syringe to ensure the entire syringe is washed, then dispenses the buffer into the wash station. The syringes are then washed at the wash station. This step is selected by default. Volume (µL). The volume of Stringent Syringe Wash Buffer to aspirate and dispense. Volumes higher than the default volume are unlikely to improve the syringe cleaning but will increase the run time. Volumes lower than the default volume may be insufficient for efficient syringe washing. • Default: 50 • Range: 0–250 Note: Setting Volume at 0 skips 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
Re-Equilibrate	This step conditions the resin and immobilized ligand to prepare them for the next step in the workflow, for example, running the Affinity Purification protocol. The Re-Equilibration step aspirates the Equilibration Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the wash station. The cartridges are parked and then the syringes are washed at the wash station. After the re-equilibration operations, this step aspirates any excess liquid remaining in the cartridge cups, dispenses the liquid at the wash station, and then performs a final syringe wash. Do not allow cartridges to dry out. After re-equilibration, use immediately for affinity purification or store them short term in the receiver plate containing 200 µL of Equilibration Buffer per well. 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. • Volume for 5 µL cartridges: – Default: 50 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 250 – Range: 0–250 Note: Setting the volume to zero will skip all Re-Equilibrate step tasks except syringe washing. Flow rate (µL/min). A flow rate slower than the default rate 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 in the cartridge resin bed. • Default: 10 • Practical: 5–20 • 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
Final Syringe Wash	Uses DI water from the wash station to flush any potential contaminants from the syringes. Each wash cycle aspirates 250 µL of fresh DI water into the syringes and dispenses the fluid at an offset between the chimneys on the wash station. Increasing the number of Wash Cycles might clean the syringes better. However, increasing the value also increases the duration of the assay and wear on the syringes. 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 iterations also increases the dilution factor, but with a cost of increased assay time and reduced syringe lifetime. Wash Cycles: • Default: 3 • Practical: 3–5 • Range: 0–10

 

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Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Immobilization protocol. Changing the selections or parameters will alter the movements.

 

Protocol step	Head moves to deck location...	Action
Starting protocol	2	Park any cartridges that may have been mounted on the head from a protocol that had been previously aborted.
	1	Dispense any liquid remaining in the syringes into the wash station.
Initial Syringe Wash	1	Wash the syringes the specified number of times.
Prime	3	Aspirate 10 µL of Priming Buffer for the cartridge air-gap prevention step.
	2	Dispense the 10 µL of buffer into the cartridge cups and exercise the cartridges off task.
	3	Aspirate the Priming Buffer.
	2	Mount the cartridges on the head.
	1	Dispense the Priming Buffer through the cartridges into the wash station.
	2	Park the cartridges in the seating station.
	1	Wash the syringes at the wash station.
Equilibrate	3	Aspirate the Equilibration Buffer.
	2	Mount the cartridges on the head.
	1	Dispense the Equilibration Buffer through the cartridges to the wash station.
	2	Park the cartridges in the seating station.
	1	Wash the syringes at the wash station.
Load Samples	4	Aspirate samples into the syringes.
	1	Wash exterior of probes at the wash station.
	2	Mount the cartridges on the head.
	7	Dispense sample through the cartridges to load sample. Collect flow-through in the Flow Through Collection plate.
	1	Wash the cartridge exteriors at the wash station.
	2	Park the cartridges at the seating station.
	1	Wash the syringes at the wash station.
Cup Wash 1	5	Aspirate Cartridge Wash Buffer 1 into the syringes.
	2	Perform the cup wash and exercise the cartridges off task.
	1	Dispense buffer at the wash station.
	1	Wash the syringes at the wash station.
Internal Cartridge Wash 1	5	Aspirate 10 µL of Cartridge Wash Buffer for the cartridge air-gap prevention step.
	2	Dispense the 10 µL of buffer into the cartridge cups and exercise the cartridges off task.
	5	Aspirate Cartridge Wash Buffer 1 into the syringes.
	2	Mount the cartridges on the head.
	1	Dispense 25 µL Cartridge Wash Buffer 1 through the cartridges at the Load Samples flow rate for the sample chase step.
	1	Dispense Cartridge Wash Buffer 1 through the cartridges for the Internal Cartridge Wash 1 step.
	1	Wash the cartridge exteriors at the wash station.
	2	Park the cartridges at the seating station.
	1	Wash the syringes at the wash station.
Load Blocking Reagent	9	Aspirate Blocking Reagent into syringes.
	1	Wash exterior of probes at the wash station.
	2	Mount the cartridges on the head.
	1	Dispense Blocking Reagent through the cartridges.
	1	Wash the cartridge exteriors at the wash station.
	2	Park the cartridges at the seating station.
	1	Wash the syringes at the wash station.
Cup Wash 2	6	Aspirate Cartridge Wash Buffer 2 into the syringes.
	2	Perform the cup wash and exercise the cartridges off task.
	1	Dispense buffer at the wash station.
	1	Wash the syringes at the wash station.
Internal Cartridge Wash 2	6	Aspirate 10 µL of Cartridge Wash Buffer 2 for the cartridge air-gap prevention step.
	2	Dispense the 10 µL of buffer into the cartridge cups and exercise the cartridges off task.
	6	Aspirate Cartridge Wash Buffer 2 into the syringes.
	2	Mount the cartridges on the head.
	1	Dispense Cartridge Wash Buffer 2 through the cartridges.
	1	Wash the cartridge exteriors 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 Stringent Syringe Wash Buffer.
	1	Dispense the buffer at the wash station.
	1	Wash the syringes at the wash station.
Re-Equilibrate	3	Aspirate the Equilibration Buffer.
	2	Mount the cartridges.
	1	Dispense Re-equilibration Buffer through the cartridges into the wash station.
	1	Wash the cartridge exteriors at the wash station.
	2	Park the cartridges at the seating station.
	1	Wash the syringes at the wash station.
	2	Aspirate excess liquid from the cartridge cups, and exercise the Cartridges Off mechanism.
	1	Dispense liquid to waste and perform a syringe wash at the wash station.
Final Syringe Wash	1	Wash the syringes at the wash station.

 

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