Assay development guidelines and protocol notes

This topic explains each step of the protocol so that you can optimize the Affinity Purification 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. This step is selected by default. 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 no or low amounts of organic solvent or known 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. This step is selected by default. 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 cartridge: – Default: 100 – Range: 0–250 • Volume for 25 µL cartridge: – 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 Prime 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. 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. • Volume for 5 µL cartridge: – Default: 50 – Range: 0–250 • Volume for 25 µL cartridge: – Default: 250 – 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 (µL/min). 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 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, 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 then 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 AssayMAP affinity purification 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 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-specific 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 the volume to zero 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 results 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. • 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. If this step is not selected, the flow-through from the Load Samples step is dispensed in 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 solution 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. 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: 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. Before the cartridge wash begins, 10 µL of Cartridge Wash Buffer 1 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 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 is 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 before the Internal Cartridge wash begins. 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 more than 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 the volume to zero 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 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 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 step 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 in the wash station. 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 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 the previous cartridge wash 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. This step is selected by default. 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 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	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. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer, 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 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. Volumes higher than the default volume may improve the purification marginally but will 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 the volume to 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 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 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 directly into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through is dispensed directly into 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 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. This step is selected by default. Volume (µL). The volume of Elution & 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 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 bound analytes from the cartridges. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Eluate Collection plate. 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, the eluate is 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. You can also select the Eluate Discard and Add to Flow Through substeps, which are described in the following rows of this table. After all the Elute operations, this step aspirates any excess liquid remaining in the cartridge cups, dispenses the liquid at the wash station, and then performs a syringe wash. This step is selected by default. Volume (µL). The maximum volume of eluate that can be collected in the Eluate Collection plate is dependent on the maximum practical working volume of the labware defined for the Affinity Purification protocol. The total volume in the Eluate Collection plate is determined by summing the net elution volume (Elute volume - Eluate Discard volume) and the Existing Collection Volume. For labware-specific maximum well volumes, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. • Volume for 5 µL cartridges: – Default: 25 – Practical: 10–30 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 125 – Practical: 50–150 – 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 (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 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 step permits a specified volume of the first eluate from the cartridges to be discarded. The Elute step aspirates the Elution Buffer into the syringes, cartridges are mounted at the seating station, and Elution Buffer is dispensed through the cartridges at the specified Elute flow rate. The Eluate Discard volume is dispensed to the wash station or the Flow Through Collection plate (if the Add to Flow Through step is selected). The remaining Elution Buffer is dispensed through cartridges into the Eluate Collection plate. Example: If the Elute, Eluate Discard, and Add to Flow Through steps are all selected with the following settings, Elute volume = 25 µL Eluate Discard volume = 5 µL the first 5 µL eluate from the cartridges will be discarded to the Flow Through Collection plate, and the remaining 20 µL eluate will be collected in the Eluate Collection plate. Select the Eluate Discard step in situations where minimizing the volume of eluate is critical. For some experimental situations, minimizing the total volume of sample eluates is critical to downstream processing. An example case is when the concentration of organic solvent must be diluted to suitable levels for reversed-phase LC/MS analyses, but the total sample volume must stay within the allowable injection volume in order to inject the entire sample. 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 initial elution volume (~2 µL for 5 µL cartridges and ~10 µL for the 25 µL cartridges) contains small 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 for each combination of sample, buffer, cartridge, and system. This option is not selected by default. Volume (µL). 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: – 5 µL cartridges: 2 – 25 µL cartridges: 10 • Range: 0–250
Add to Flow Through	A substep of the Elute and Eluate Discard steps, if selected the Add to Flow Through step dispenses the Eluate Discard volume to the Flow Through Collection plate. If not selected, the Eluate Discard is dispensed to the wash station. This option is not selected by default.
Existing Collection Volume	This step enables you to specify an amount of liquid that is already contained within the wells of the Eluate Collection plate. The Existing Collection Volume and the net volume from the Elute step (Elute volume - Eluate Discard volume) 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, 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, for immediately adjusting the pH of the eluates, or to aid in the recovery of small volumes of eluates from AssayMAP cartridges. Volume (µL): • Default: 0 • Range: 0—300
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 iterations also increases the dilution factor, but with a cost of increased assay time and reduced syringe lifetime. This step is selected by default. 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 Affinity Purification protocol using the default protocol settings. Changing the selections or parameters will alter the movements.

 

Protocol step	Head moves to deck location...	Action
Start protocol	2	Parks any cartridges that may have been mounted 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 the specified number of times.
Prime	3	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.
	3	Aspirates the Priming Buffer.
	2	Mounts the cartridges on the head.
	1	Dispenses the Priming Buffer through the cartridges into the wash station.
	2	Parks the cartridges in the seating station.
	1	Washes the syringes at the wash station.
Equilibrate	3	Aspirates the Equilibration Buffer.
	2	Mounts the cartridges on the head.
	1	Dispenses the Equilibration Buffer through the cartridges to equilibrate.
	2	Parks the cartridges in the seating station.
	1	Washes the syringes at the wash station.
Load Samples	4	Aspirates samples into the syringes.
	1	Washes the exterior of the syringe probes at the wash station.
	2	Mounts the cartridges on the head.
	7	Dispenses sample through the cartridges to load sample. Collect flow-through in the Flow Through Collection plate.
	1	Washes the cartridge exteriors at the wash station.
	2	Parks the cartridges at the seating station.
	1	Washes the syringes at the wash station.
Cup Wash 1	5	Aspirates Cartridge Wash Buffer 1 into the syringes.
	2	Performs the cup wash and exercises the cartridges off task.
	1	Dispenses the buffer at the wash station.
	1	Washes the syringes at the wash station.
Internal Cartridge Wash 1	5	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.
	5	Aspirates Cartridge Wash Buffer 1 into the syringes.
	2	Mounts the cartridges on the head.
	1	Dispenses 25 µL Cartridge Wash Buffer 1 through the cartridges at the Load Samples flow rate for the sample chase step.
	1	Dispenses Cartridge Wash Buffer 1 through the cartridges for the Internal Cartridge Wash step.
	1	Washes the exterior of the cartridge tips at the wash station.
	2	Parks the cartridges at the seating station.
	1	Washes the syringes at the wash station.
Cup Wash 2	6	Aspirates Cartridge Wash Buffer 2 into the syringes.
	2	Performs a cup wash and exercises the cartridges off task.
	1	Dispenses buffer at the wash station.
	1	Washes the syringes at the wash station.
Internal Cartridge Wash 2	6	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.
	6	Aspirates Cartridge Wash Buffer 2 into the syringes.
	2	Mounts the cartridges on the head.
	1	Dispenses Cartridge Wash Buffer 2 through the cartridges.
	1	Washes the exterior of the cartridge tips at the wash station.
	2	Parks the cartridges at the seating station.
	1	Washes the syringes at the wash station.
Stringent Syringe Wash	8	Aspirates the Stringent Syringe Wash Buffer (Elution Buffer).
	1	Dispenses the buffer at the wash station.
	1	Washes the syringes at the wash station.
Elute	8	Aspirates the Elution Buffer.
	2	Mounts the cartridges.
	9	Elutes the samples into the Eluate Collection plate.
	1	Washes the cartridge exteriors at the wash station.
	2	Parks the cartridges at the seating station.
	9	Mixes eluates.
	1	Washes the syringes at the wash station.
	2	Aspirates excess liquid from the cartridge cups, and exercise the Cartridges Off mechanism.
	1	Dispenses liquid to waste and perform a syringe wash at the wash station.
Final Syringe Wash	1	Washes the syringes at the wash station.

 

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