Assay development guidelines and protocol notes
This topic explains the following:
• Each step of the protocol so that you can optimize the Fractionation 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 or 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. 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. In preparation for priming, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the Organic Waste plate, 10 µL of Priming Buffer is aspirated 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. The Prime step aspirates the Priming Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Organic Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station. This step is selected by default. Volume (µL). The default volume is sufficient to wet and remove entrained air from the resin bed. 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 – Practical: 100–250 – Range: 0–250 • Volume for 25 µL cartridge: – Default: 250 – Practical: 250 – Range: 0–250 Note: Setting the volume to zero skips all Prime tasks except syringe washing. Flow rate (µL/min). A flow rate slower than the default value diminishes the ability to effectively remove entrained air from the resin bed. A flow rate faster than the default is not required and has not been tested. • Default: 300 • Practical: 300 • Range: 0.5–300 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: 1 • Practical: 1–3 • Range: 0–10 |
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. In preparation for equilibration, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the Organic Waste plate, 10 µL of Equilibration Buffer is aspirated 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. During the Equilibrate step, the Equilibration Buffer is aspirated into the syringes, the cartridges are mounted, and then the buffer is dispensed through the cartridges into the Organic Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station. The AssayMAP reversed-phase cartridges (C18 and RP-S), which are often used with the Fractionation application, require an equilibration solution that has a very low concentration or no organic solvent for effective binding during the sample loading step. 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 – Practical: 50–100 – Range: 0–250 • Volume for 25 µL cartridge: – Default: 250 – Practical: 250 – Range: 0–250 Note: Setting the volume to zero skips all Equilibrate 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 may not equilibrate through the pores in the beads. • Default: 10 • Practical: 5–20 • Range: 0.5–500 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: 1 • Practical: 1–3 • Range: 0–10 |
Load Samples | This step allows the target analytes to bind to the surface chemistry of the resin bed. No liquid is removed or added to the cartridge cups before the sample loading begins. The assumption is that there is still liquid in the cups from the equilibration step that will prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. This step aspirates sample into the syringes and then mounts the cartridges. The samples are dispensed through the cartridges into the Flow Through Collection plate or Organic Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any sample on the exterior of the cartridges, the cartridges are parked at the seating station, and the syringes are washed at the wash station.  Be sure to include the recommended labware-specific volume overage to prevent air from entering the cartridge. For details, 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. • Default: 100 • Practical: 10–250 • Range: 0–250 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: – 2–10 (5 µL cartridges) – 5–20 (25 µL cartridges) • 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 Organic Waste plate. The Collect Flow Through step is skipped if the Load Samples step is not conducted. This step is selected by default. |
Cup Wash | This step removes the residual sample solution that may remain above the resin bed after the Load Samples step. The Cup Wash step aspirates Wash Buffer 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 Organic Waste plate, and then washing the syringes at the wash station. This step is selected by default for the 5 µL cartridges. The Cup Wash and Internal Cartridge Wash steps are not an option for the 25 µL cartridges because of the volume limit of the wells in the labware at deck location 9. Instead, a cartridge wash should be done in place of one of the elution steps for the 25 µL cartridges. 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 | This step uses Wash Buffer to wash non-specifically bound molecules from the resin bed. In preparation for the Internal Cartridge Wash, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the Organic Waste plate, 10 µL of Wash Buffer is aspirated 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 Wash Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or Organic Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. If the Load Samples step is selected, the first 5 µL (5 µL cartridges) of Wash Buffer is dispensed as a sample chase at the Load Samples flow rate. Next, the Internal Cartridge Wash volume minus the chase volume is dispensed at the Internal Cartridge Wash 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. This step is selected by default for the 5 µL cartridges. The Cup Wash and Internal Cartridge Wash steps are not an option for the 25 µL cartridges because of the volume limit of the wells in the labware at deck location 9. Instead, a cartridge wash should be done in place of one of the elution steps for the 25 µL cartridges. 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 for 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 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 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 flow-through from Internal Cartridge Wash step is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from Internal Cartridge Wash is dispensed into the Organic Waste plate. This step is selected by default. This step is skipped if the Internal Cartridge Wash step is not conducted. |
Predispense Elution Buffer | This step maximizes the recovery of small volume elutions (< ~ 20 µL) by allowing direct elution into a liquid. Small volumes may cling to the end of the cartridge during elution that cannot be removed by the programmed tip touches in the wells of the plates. This step transfers a specified volume of Elution Buffer from the appropriate Elution Buffer plate (1-6) to the corresponding Fraction Collection plate before eluting each fraction into the Fraction Collection plate. Volume (µL): • Default: 15 • Range: 0–50 |
Elute Fraction 1–6 | Before running the Fractionation protocol, the Stack Plates protocol stacks a set of Elution Buffer plates at deck location 4 and a set of Fraction Collection plates at deck location 7. The number of Fraction Collections plates is equal to the Number of Fractions you specified. In preparation for elution, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the wash station, 10 µL of Elution Buffer is aspirated 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. During this step, the plate movements on the deck are as follows: 1 The Sample plate and Flow Through Collection plate move one position to the right and are stacked on the Priming Buffer plate and Equilibration Buffer plate, respectively. 2 To collect fractions 1-4, the Elution Buffer and Fraction Collection plates are moved from the stacks a locations 4 and 7 to deck locations 5 and 8 before each elution step. 3 The Elution Buffer and Fraction Collection plates for fractions 1–4 are stacked at deck locations 6 and 9 after each elution step. For example: Elution Buffer plate 1 is moved from the Elution Buffer plates stacked at location 4 to location 5. Fraction Collection plate 1 is moved from the Fraction Collection plates stacked at location 7 to location 8. Elution buffer is aspirated into the syringes from Elution Buffer Plate 1 at location 5. If the Predispense Elution Buffer step was selected, this volume will be dispensed to Fraction Collection plate 1 at location 8. Cartridges are mounted and samples are eluted from the cartridge directly into Fraction Collection plate 1. The cartridges are parked and the syringes are washed at the wash station. Fraction Collection plate 1 is moved from location 8 and stacked on location 9. Elution Buffer plate 1 is moved from location 5 and stacked on location 6. |
4 For fraction 5, step 2 is conducted but instead of stacking the Elution Buffer and Fraction Collection plates (step 3), they remain at locations 5 and 8, respectively. 5 For fraction 6, there is no plate movement. Elution Buffer plate 6 remains at location 4 and Fraction Collection plate 6 remains at location 7. Final Fraction Collection plates 1-4 will be in deck location 9, Fraction Collection plate 5 will be in deck location 8 and Fraction Collection plate 6 will be in deck location 7. 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, or 50–75 µL for 25 µL cartridges). The default volumes are conservative and significantly higher than the minimum expected with a strong Elution Buffer. • 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 skips all Elute tasks except syringe washing. 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 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 | |
Final Syringe Wash | This step uses the wash station to flush potential contaminants from the syringes. Before the final syringe wash begins, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the Organic Waste plate. No solution is added into the cartridge cups. Note: If the Final Syringe Wash is skipped, the 10 µL of elution buffer will remain in the cartridge cups. 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 and reduced syringe lifetime. 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 |
Automation movements during the protocol
Stack Plates protocol
This section describes the basic movements of the AssayMAP Bravo Platform during the Stack Plates protocol. Changing the Number of Fractions will alter the movements.
Protocol step | Head moves to deck location... | Action |
|---|---|---|
Starting protocol | 4 | Checks the labware height. |
7 | Checks the labware height. | |
Stack Elution Buffer plates | 8 to 4 | Moves the Elution Buffer plate from deck location 8 to 4. |
5 to 4 9 to 4 6 to 4 3 to 4 | Moves the remaining Elution Buffer plates, in the order listed, to deck location 4. | |
Stack Fraction Collection plates | 8 to 7 | Moves the Fraction Collection plate from deck location 8 to 7. |
5 to 7 9 to 7 6 to 7 3 to 7 | Moves the remaining Fraction Collection plates, in the order listed, to deck location 7. |
Fractionation protocol
This section describes the basic movements of the AssayMAP Bravo Platform during the Fractionation protocol using the default method. Changing the selections or parameters will alter the movements.
Protocol step | Head moves to deck location... | Action |
|---|---|---|
Starting protocol | 4 | Checks the Elution Buffer plate stack height. |
7 | Checks the Fraction Collection plate stack height. | |
9 | Checks the labware height. | |
6 | Checks the labware height. | |
2 | Parks all cartridges that might have been loaded on the head from a previously aborted protocol. | |
1 | Dispenses any liquid remaining in the syringes into the wash station. | |
Initial Syringe Wash | 1 | Washes the syringes. |
Prime Cartridges | 2 | Aspirates 20 µL of air above this location, moves down to just above the cartridge engagement point and aspirates 60 µL, and then exercises the cartridges off task. |
3 | Dispenses into the Organic Waste plate. | |
1 | Performs an external probe wash. | |
6 | 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. | |
6 | Aspirates the Priming Buffer. | |
2 | Mounts the cartridges on the head. | |
3 | Dispenses the Priming Buffer through the cartridges and into Organic Waste plate. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
Equilibrate Cartridges | 2 | Aspirates 20 µL of air above this location, moves down to just above the cartridge engagement point and aspirates 60 µL, and then exercises the cartridges off task. |
3 | Dispenses into the Organic Waste plate. | |
1 | Performs an external probe wash. | |
9 | Aspirates 10 µL of Equilibration Buffer for the cartridge air-gap-prevention step. | |
2 | Dispenses the 10 µL of Equilibration Buffer into the cartridge cups and exercises the cartridges off task. | |
9 | Aspirates the Equilibration Buffer. | |
2 | Mounts the cartridges on the head. | |
3 | Dispenses the Equilibration Buffer through the cartridges into the Organic Waste plate. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
Load Samples | 5 | Aspirates the samples into the syringes. |
2 | Mounts the cartridges on the head. | |
8 | Dispenses the samples through the cartridges and into the Flow through Collection plate. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
Cup Wash (5 µL cartridges only) | 9 | Aspirates the Wash Buffer. |
2 | Washes the cartridge cups and exercises the cartridges off task. | |
3 | Dispenses the Wash Buffer into the Organic Waste plate. | |
1 | Washes the syringes. | |
Internal Cartridge Wash (5 µL cartridges only) | 2 | Aspirates 20 µL of air above this location, moves down to just above the cartridge engagement point and aspirates 60 µL, and then exercises the cartridges off task. |
3 | Dispenses into the Organic Waste plate. | |
1 | Performs an external probe wash. | |
9 | Aspirates 10 µL of Wash Buffer for the cartridge air-gap-prevention step. | |
2 | Dispenses the 10 µL of Wash Buffer into the cartridge cups and exercises the cartridges off task. | |
9 | Aspirates the Wash Buffer. | |
2 | Mounts cartridges on the head. | |
3 | Dispenses 5 µL Wash Buffer through the cartridges at the Load Samples flow rate for the sample chase step. | |
3 | Dispenses the remaining Wash buffer through the cartridges at the Internal Cartridge Wash flow rate and into the Organic Waste plate. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
5 | Moves the Sample plate to deck location 6. | |
8 | Moves the Flow Through Collection plate to deck location 9. | |
Elute Fractions 1–6 | 4 to 5 | Moves Elution Buffer plate from deck location 4 to 5. |
7 to 8 | Moves Fraction Collection plate from deck location 7 to 8. | |
5 | Aspirates the Elution Buffer for elution. | |
2 | Mounts the cartridges on the head. | |
8 | Elutes into the Fraction Collection plate. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
8 to 9 | Moves Fraction Collection plate from deck location 8 to 9. | |
5 to 6 | Moves Elution Buffer plate from deck location 5 to 6. | |
Repeats for fractions 2, 3, and 4. | ||
Repeats for fraction 5, except the final plates remain at deck locations 5 and 8. | ||
Repeats for fraction 6, except the plates remain at deck locations 4 and 7. Note: Final Fraction Collection plates 1-4 will be at deck location 9. Fraction Collection plate 5 will be in deck location 8. Fraction Collection plate 6 will be at deck location 7. | ||
Final Syringe Wash | 2 | Aspirates 20 µL of air above this location, moves down to just above the cartridge engagement point and aspirates 60 µL, and then exercises the cartridges off task. |
3 | Dispenses into the Organic Waste plate. | |
1 | Washes the syringes. | |