Assay development guidelines and protocol notes

This topic explains each step of the Affinity Purification: 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 Cartridges	This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that this selection is correct. If the cartridge selection is: • Greater than the actual number of used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the form specifies 96 cartridges, but only 8 cartridges are in the seating station, the head will apply 12 times more force than what is required. • Less than the actual number used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the form specifies 8 cartridges, but 96 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. To obtain expected instrument performance, ensure that the cartridge selection is correct. IMPORTANT Although you specify the number of cartridges for this app, you specify the number of columns of cartridges for other apps. Default: 8 Range: 1-12
Initial Syringe Wash	Flushes potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the 250 µL are aspirated into the syringes from the wash station chimneys, and then the head moves by a fixed offset between the chimneys to dispense the liquid 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: 5 • Practical: 3–5 • Range: 1–10
Prime	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. During this step, the syringes fill with sufficient Priming & Equilibration Buffer to wet the inlet seal (10 μL), and perform the Prime (100 μL) and Equilibration steps (50 μL default). Before mounting the cartridges on the probes, the Bravo 96AM head dispenses 10 μL into each cartridge cup to fill the small space between the syringe probes and the upper cartridge membrane. This prewetting of the inlet seal ensures that no air bubbles remain trapped in this small area and leaves a visible amount of liquid in the cartridge cup for the duration of the run. After mounting the cartridges on the probes, the head moves between the chimneys at the wash station to prime the cartridges by dispensing 100 μL at 300 μL/min through the cartridges to waste. This step is selected by default.
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 dispenses the Equilibration Buffer remaining in the syringes through the cartridges. The step ensures that the resin bed has been fully equilibrated by using a slower buffer loading flow rate than the Prime 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. • Default: 50 • Practical: 50–100 • Range: 1–140 If you select Prime and Equilibrate, the system calculates the volume required to prewet the cartridges and perform both steps and then aspirates that volume during the Prime step. 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. • Default: 25 • Practical: 5–25 • Range: 0.5–500
Load Samples	Loads samples onto the cartridges by aspiration. This step mounts the cartridges on the syringe probes, aspirates the sample through the resin bed, and performs an external cartridge wash. A 5 µL sample chase of Priming & Equilibration Buffer is aspirated through the resin bed at the same flow rate as the sample load to ensure that the sample in the resin bed at the end of the sample load has the same residence time for binding as the rest of the sample. An exterior cartridge wash is performed, and then the contents in the syringe (flow-through) are deposited either in the Flow Through Collection plate if Collect Flow Through is selected, or in the wash station. The protocol accommodates sample volumes up to 1000 µL to be aspirated 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 of times to load and the volume of each iterative load, 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. 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: 25–250 • Range: 0–1000
	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 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 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 selected, this step dispenses the sample flow-through from the Load Sample step in the Flow Through Collection plate. If the Collect Flow Through step is not selected (default), the flow-through from the Load Sample step is dispensed in the wash station at an offset between the chimneys. The Collect Flow Through step is skipped if the Load Sample step is not conducted. This step is not selected by default. Note: A constant 5-µL volume is used as a sample chase during the Load Samples step, which results in some dilution of sample flow-through. For example, if you load a 50-µL sample, the flow-through will contain the volume of the sample plus 5 µL of sample chase. Make sure you consider this volume when calculating the flow-through concentration.
Cup Wash 1	Removes the residual sample liquid that may remain above the upper cartridge membrane after the Load Samples step. The Cup Wash 1 step aspirates 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 get stuck to the probes during the cup wash are parked at the seating station, and then the liquid in the syringes is dispensed into the wash station at an offset from the chimneys. The syringes are then 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: 50 • Practical: 25–50 • Range: 1–100 Wash cycle. Each cycle comprises one cup wash and one syringe wash. • Default: 2 • Practical: 1–3 • Range: 1–10 Each wash cycle consists of a cup wash followed by syringe wash.
Internal Cartridge Wash 1	Uses Cartridge Wash Buffer 1 to wash non-specifically bound molecules from the cartridge resin. This step aspirates Cartridge Wash Buffer 1 through the mounted cartridges into the syringes, parks the cartridges at the seating station, and then dispenses the syringe contents into the wash station at an offset from the chimneys. The syringes are then washed at the wash station. 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 will also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. • Default: 50 • Practical: 50–100 • Range: 1–250 Flow rate (µL/min). A rate slower than 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: 25 • 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: 5 • Practical: 2–5 • Range: 0–10
Cup Wash 2	Removes the residual sample liquid or buffer that may remain above the upper cartridge membrane after the Internal Cartridge Wash 1 step. This step aspirates Cartridge Wash Buffer 2 into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from the previous cartridge wash is aspirated from the cartridge cups. Any cartridges that get stuck to the probes during the cup wash are parked at the seating station, and then the liquid in the syringes is dispensed into the wash station at an offset from the chimneys. The syringes are then washed at the wash station. This step is not 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: 50 • Practical: 25–50 • Range: 1–100 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: 1 • Practical: 1–3 • Range: 1–10
Internal Cartridge Wash 2	Uses Cartridge Wash Buffer 2 to wash non-target elements from the cartridge resin. This step aspirates Cartridge Wash Buffer 2 through the mounted cartridges into the syringes, parks the cartridges at the seating station, and then dispenses the buffer into the wash station at an offset from the chimneys. The syringes are then washed at the wash station. Select the Internal Cartridge Wash 2 step to provide an additional washing step to minimize non-target elements from the sample matrix in the cartridge, which could possibly contaminate the eluate. This step is not 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. • Default: 50 • Practical: 50–100 • Range: 1–250 Flow rate (µL/min). A rate slower than 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: 25 • 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 DI water is used for each syringe wash cycle. • Default: 5 • Practical: 2–5 • Range: 0–10
Stringent Syringe Wash	Cleans the syringes with the Elution Buffer prior to elution. The Stringent Syringe Wash step aspirates the Elution Buffer into the syringes, and then dispenses the buffer into the wash station at an offset between the chimneys. The syringes are then washed at the wash station. This step is selected by default. Volume (µL/min). 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 increase run time. Volumes lower than the default volume may be insufficient for efficient syringe washing. • Default: 50 • Practical: 50–100 • Range: 1–250 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: 5 • Practical: 1–3 • Range: 1–10
Elute	Uses Elution & Syringe Wash Buffer to elute bound analytes from the cartridges. This step aspirates the 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. This step is selected by default. Volume (µL). Using sufficiently strong elution conditions and the default flow rate (5 µL/min), targets can typically be fully eluted from cartridges in approximately two cartridge column volumes. • Default: 50 • Practical: 10–30 • Range: 1–250 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
Re-Equilibrate	Uses the Priming & Equilibration Buffer to return the cartridge to a neutral pH solution. This step aspirates Priming & Equilibration Buffer through the mounted cartridges into the syringes, parks the cartridges, and then dispenses the liquid into the wash station between the chimneys. The cartridges are parked, the syringes are washed, and then the syringes are used to mix the samples in the Eluate Collection plate. Note: If the total volume in the Eluate Collection plate is <15  µL, the samples are not mixed. Select Re-Equilibrate after eluting the sample to displace residual elution buffer (low pH) and return the cartridge pH to a neutral level if the cartridges will be reused or stored. 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: 1–250 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 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
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 in the wash station. Note: The amount of carryover in the syringe (expressed as dilution of the starting sample) depends on the total number of syringe wash cycles. Three cycles give a dilution of up to 103. 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: 5 • Practical: 3–5 • Range: 1–10

Return to top

Minimizing the duration of your assay

To minimize the duration of your assay:

If the sum of the sample load, chase, and both internal cartridge washes is less than 250 μL, modify the following protocol settings to minimize the Bravo Platform actions:

•	Load Samples: Set the number of wash cycles to 0.

•	Collect Flow Through: Not selected.

•	Cup Wash 1: Not selected.

•	Internal Cartridge Wash 1: Set the number of wash cycles to 0. (If Internal Cartridge Wash 2 is not selected, set the number of wash cycles to any desired value.)

•	Cup Wash 2: Not selected.

These settings result in accumulation of the flow-through, chase, and any internal cartridge wash liquids in the syringe without requiring removal of the cartridges from the probes. This total volume must be less than the 250 μL syringe capacity, as the cartridges will dismount for larger accumulated volumes and introduce the potential for contamination of the cup, which would make skipping the cup washes a poor choice. The software keeps track of cumulative volume of the sample (which equals the flow-through), 5-μL chase, and Internal Cartridge Wash 1 and 2 drawn into the syringe, and performs the steps required to empty the syringe when it reaches the 250-μL capacity. As long as the accumulated volume is less than 250 μL, this volume can be held in the syringes until the Stringent Syringe Wash initiates.

Return to top

Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Affinity Purification: Aspiration Mode 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 the Priming & Equilibration Buffer for the Prime and Equilibrate steps.
	2	Dispenses 10 µL of buffer into each cartridge cup.
	2	Mounts the cartridges on the syringe probes.
	1	Dispenses the buffer for the Prime step through the cartridges into the wash station.
Equilibrate	1	Dispenses the remaining buffer through the cartridges into the wash station at the specified flow rate.
Load Samples	4	Aspirates samples through the mounted cartridges into the syringes.
	1	Washes the exterior tips of the cartridges at the wash station.
	3	Aspirates a 5 µL sample chase.
	2	Parks the cartridges at the seating station.
	7	Dispenses the sample flow-through into the wash 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	2	Mounts the cartridges on the syringe probes.
	5	Aspirates Cartridge Wash Buffer 1 through the mounted cartridges.
	1	Washes the exterior of the cartridges at the wash station.
	2	Parks the cartridges at the seating station.
	1	Dispenses the buffer at the wash station.
	1	Washes the syringes at the wash station.
Stringent Syringe Wash	8	Aspirates Elution & Syringe Wash Buffer.
	1	Dispenses the buffer at the wash station.
		Washes the syringes at the wash station.
Elute	8	Aspirates the Elution & Syringe Wash Buffer.
	2	Mounts the cartridges on the syringe probes.
	9	Elutes the samples into the Eluate Collection plate.
	1	Washes the cartridge exteriors.
Re-Equilibrate and Eluate Mix	3	Aspirates Equilibration Buffer through the cartridges into the syringes.
	1	Washes the cartridge exteriors at the wash station.
	2	Parks the cartridges at the seating station.
	1	Washes the syringes.
	9	Mixes the samples into the Eluate Collection plate using the syringes.
	1	Washes the syringes.
Final Syringe Wash	1	Washes the syringes at the wash station.

 

Return to top