Assay development guidelines and protocol notes
This topic explains the following:
• Each step of the protocol so that you can optimize the Affinity Purification: Aspiration Mode protocol to your particular experimental design
• Automation movements during the protocol
For details on how to use the Experiments Editor, see Using Experiment IDs.
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 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 software 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.  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 | 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. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. • Default: 3 • Practical: 3–5 • Range: 0–10 |
Prime | This step removes entrained air from the packed resin bed and properly wets the surface of the resin. The Prime step aspirates enough Priming Buffer into the syringes to wet the inlet seal (10 μL), and perform the Prime (100 μL) and Equilibration (50 μL default) steps. In preparation for priming, 10 μL of Priming Buffer is dispensed into each cartridge cup to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. After mounting the cartridges on the probes, the Priming Buffer is dispensed (100 μL at 300 μL/min) through the cartridges into the wash station. 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. This step is selected by default. Volume (µL). The volume is sufficient to wet and remove entrained air from the resin bed. • Default: 100 • Range: 100 Flow rate (µL/min). • Default: 300 • Range: 300 |
Equilibrate | This step ensures that the resin bed is fully equilibrated with a solution that provides the optimal chemical conditions for binding during the Load Samples step. This step dispenses the Equilibration Buffer remaining in the syringes through the cartridges. This step is selected by default. Volume (µL). The default volume is equal to 10 column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. • Default: 50 • Practical: 50–100 • Range: 0–140 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 the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 µL/min may not equilibrate through the pores in the beads. • Default: 10 • Practical: 5–20 • Range: 0.5–500 |
Load Samples | This step allows the target analytes to bind to the surface chemistry of the resin bed. This step mounts the cartridges on the syringe probes, aspirates the sample through the resin bed, and washes the exterior of the cartridge tips at the wash station to remove any sample on the outside of the cartridge tips. 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, cartridges are ejected, and then the contents in the syringe (flow-through) are deposited into the Flow Through Collection plate or 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. 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: 100 • Practical: 10–1000 • Range: 0–1000 Note: The lower the sample volume the higher the percentage of the total volume is overage. To minimize sample loss, Agilent recommends diluting small volume samples. Note: Setting the volume to zero skips all Load Samples tasks except 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. Therefore, the maximum possible quantitative binding capacity is only obtained with very slow sample loading flow rates. If the amount of sample that you want to capture is significantly lower than the total possible qualitative binding capacity, you will be able to use a faster flow rate while maintaining quantitative binding. Using flow rates slower than the default may not significantly increase analyte binding, and using flow rates faster than the default will decrease the quantitative binding capacity of the cartridges. • Default: 5 • Practical: 2–15 • Range: 0.1–500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 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 into the Flow Through Collection plate. If this step is not selected, the flow-through from the Load Samples step is dispensed into the wash station. The Collect Flow Through step is skipped if the Load Samples 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 | This step removes the residual sample solution that may remain above the resin bed after the Load Samples step. The Cup Wash 1 step aspirates Cartridge Wash Buffer 1 into the syringes 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. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the wash station, and then washing the syringes at the wash station. This step is selected by default. Volume (µL). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 µL may offer little benefit. • Default: 25 • Practical: 25–50 • Range: 0–100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. • Default: 3 • Practical: 3–5 • Range: 0–10 |
Internal Cartridge Wash 1 | This step uses Cartridge Wash Buffer 1 to wash non-specifically bound molecules from the resin bed. For the wash operation, 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). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increases the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. • Default: 50 • Practical: 50–100 • Range: 0–250 Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (µL/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 µL/min may 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 this step. 250 µL of DI water is used for each syringe wash cycle. • Default: 3 • Practical: 2–5 • Range: 0–10 |
Cup Wash 2 | This step removes the residual buffer that may remain above the resin bed 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 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. The syringes are washed at the wash station. This step is selected by default. Volume (µL). A volume less than the default 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 skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. • Default: 3 • Practical: 3–5 • Range: 0–10 |
Internal Cartridge Wash 2 | This step uses Cartridge Wash Buffer 2 to wash non-specifically bound molecules and Cartridge Wash Buffer 1 from the resin bed. For the wash operation, 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 washed at the wash station. This step is selected by default. Volume (µL). Volumes higher than the default volume (10 column volumes) 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: 0–250 Note: Setting the volume to 0 skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (µL/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 µL/min may 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 this step. 250 µL of DI water is used for each syringe wash cycle. • Default: 3 • Practical: 2–5 • Range: 0–10 |
Stringent Syringe Wash | This step cleans the syringes with the Elution Buffer prior to elution. The Stringent Syringe Wash step aspirates the Elution Buffer into the syringes, draws the buffer through a full syringe stroke to ensure the entire syringe is rinsed, and 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/min). Volumes higher than the default volume may improve the syringe cleaning but will increase the reagent consumption. Volumes lower than the default volume may be insufficient for efficient syringe washing. • Default: 50 • Practical: 50–100 • Range: 0–250 Note: Setting the volume to zero skips all Stringent Syringe Wash tasks. Wash cycle. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. • Default: 2 • Practical: 2–5 • Range: 0–10 |
Elute | This step uses Elution 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). The volume of Elution Buffer required for complete elution of bound analyte from the resin bed is dependent on the strength of the Elution Buffer. So the minimum elution volume must be determined empirically. If a strong Elution Buffer is used, the minimum volume is approximately 2–3 column volumes (10–15 µL for 5 µL cartridges). The default volume is conservative and significantly higher than the minimum expected with a strong Elution Buffer. • Default: 25 • Practical: 10–30 • Range: 0–250 Flow rate (µL/min). A flow rate slower than the default 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: 5–15 • Range: 0.1–500 |
Re-Equilibrate | Uses the Equilibration Buffer to return the cartridge to a neutral pH solution. During the Re-Equilibrate step, the Equilibration Buffer is aspirated through the mounted cartridges into the syringes. The cartridges are parked at the seating station and then the liquid is dispensed into the wash station between the chimneys. 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. This step is selected by default. Note: If the Re-Equilibrate step is skipped, the eluate will not be mixed. Volume (µL). The default volume is equal to 10-column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. • Default: 50 • Practical: 50–100 • Range: 0–250 Note: Setting the volume to zero skips all Equilibrate tasks. 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 may not equilibrate through the pores in the beads. • Default: 10 • Practical: 5–20 • Range: 0.5–500 |
Final Syringe Wash | This step uses the wash station to flush potential contaminants from the syringes. During each Final Syringe Wash cycle, the head aspirates 250 µL into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: • Default: 3 • Practical: 3–5 • Range: 0–10 |
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.
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 method. 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 air-gap prevention and the Prime and Equilibrate steps. |
2 | Dispenses 10 µL of buffer into each cartridge cup for air-gap prevention. | |
2 | Mounts the cartridges on the syringe probes. | |
1 | Dispenses the buffer for the Prime step through the cartridges into the wash station at the priming flow rate. | |
Equilibrate | 1 | Dispenses the remaining buffer through the cartridges into the wash station at the Equilibration flow rate, and then washes the exterior of the cartridges. |
Load Samples | 4 | Aspirates samples through the mounted cartridges into the syringes. |
1 | Washes the exterior of the cartridges. | |
3 | Aspirates a 5 µL of Priming & Equilibration Buffer for the sample chase. | |
1 | Washes the exterior of the cartridges. | |
2 | Parks the cartridges at the seating station. | |
1 | Dispenses the sample flow-through into the wash station. | |
1 | Washes the syringes. | |
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. | |
2 | Parks the cartridges at the seating station. | |
1 | Dispenses the buffer into the wash station. | |
1 | Washes the syringes. | |
Cup Wash 2 | 6 | Aspirates Cartridge Wash Buffer 2 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 2 | 2 | Mounts the cartridges on the syringe probes. |
6 | Aspirates Cartridge Wash Buffer 2 through the mounted cartridges. | |
1 | Washes the exterior of the cartridges. | |
2 | Parks the cartridges at the seating station. | |
1 | Dispenses the buffer into the wash station. | |
1 | Washes the syringes. | |
Stringent Syringe Wash | 8 | Aspirates Syringe Wash Buffer. |
1 | Dispenses the buffer into the wash station. | |
1 | Washes the exterior of the syringes. | |
Elute | 8 | Aspirates the Elution Buffer. |
2 | Mounts the cartridges on the syringe probes. | |
9 | Elutes the samples into the Eluate Collection plate. | |
1 | Washes the exterior of the cartridge tips. | |
Re-Equilibrate and Eluate Mix | 3 | Aspirates Equilibration Buffer through the cartridges into the syringes. |
2 | Parks the cartridges at the seating station. | |
1 | Dispenses the buffer, and then washes the syringes. | |
9 | Mixes the samples in the Eluate Collection plate using the syringes. | |
Final Syringe Wash | 1 | Washes the syringes. |