Assay development guidelines and protocol notes
This topic explains the following:
• Each step of the protocol so that you can optimize the Peptide Cleanup 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 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 the syringe contents 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 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. The AssayMAP reversed-phase cartridges (C18, RPS, and RPW) used with the Peptide Cleanup application require a Priming Buffer containing at least 25% organic solvent. As illustrated in Agilent app note 5991-6478EN (Reference library), reversed-phase cartridges (C18, RPS, and RPW) that are primed with solutions containing a low percentage of organic solvent have greatly reduced binding capacity. Agilent recommends using a priming solution that contains at least 50% of an organic solvent such as acetonitrile. 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 cartridges: – Default: 100 – Practical: 100–250 – Range: 0–250 • Volume for 25 µL cartridges: – 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 lower 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: 300 • 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 |
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, RPS, and RPW) typically used with the Peptide Cleanup application require an equilibration solution that has a very low concentration of 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 cartridges: – Default: 50 – Practical: 50–100 – Range: 0–250 • Volume for 25 µL cartridges: – 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 using the default volume 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 performs an external syringe wash at the wash station to remove any sample remaining on the outside of the probes before mounting 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. The protocol accommodates sample volumes up to 1000 µL to be dispensed through AssayMAP peptide cleanup 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. To determine the number and the volume of iterative loads, the protocol uses the following formulas: • # of times to load = Total sample volume/250, and the result 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-specific volume overage to prevent air from entering the cartridge. For more information, see Transferring the samples to the microplate.To determine the volume of sample to load, see Determining the volume of sample to load. 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. Large sample volumes (> 250 µL) may require slightly more excess sample due to evaporation. • 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: – 2–10 (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 in the Flow Through Collection plate. If this step is not selected, the flow-through from the Load Samples step is dispensed in 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 liquid that may remain above the resin bed after the Load Samples step. The Cup Wash step aspirates Cartridge Wash Buffer into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from the 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. 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 Cycles: Each wash cycle comprises one cup wash and one syringe wash. • Default: 3 • Practical: 3–5 • Range: 0–10 |
Internal Cartridge Wash | This step uses Cartridge 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 Cartridge 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 the Cartridge 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) or 25 µL (25 µL cartridges) of Cartridge 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. 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. • Volume for 5 µL cartridges: – Default: 50 – Practical: 50–100 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 250 – Practical: 250 – 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 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 flow-through from the 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 the Internal Cartridge Wash step is dispensed into the Organic Waste plate. This step is not selected by default. |
Stringent Syringe Wash | This step cleans the syringes with the Syringe Wash Buffer prior to elution. The Stringent Syringe Wash step aspirates the Syringe Wash 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 Organic Waste plate. The syringes are then washed at the wash station. This step is selected by default. Volume (µL). Volumes higher than the default volume are unlikely to 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 Cycles. 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 peptides from the cartridges. 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 Organic Waste plate, 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. 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 samples are mixed in the Eluate Collection plate and the syringes are washed at the wash station. 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. 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, 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. Note: The Eluate Collection plate must be able to accommodate the total volume, which 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 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 buffer may be required to get the same elution yield at high flow rates relative to using lower flow rates for a given elution volume). • Default: 5 • Practical: 5–15 • 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: 1 • Practical: 1–3 • Range: 0–10 |
Eluate Discard | This substep of the Elute step permits a specified volume of the eluate from the cartridges to be discarded before the eluate starts to be collected during the Elute step. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the Elution Buffer at the Elute flow rate through the cartridges. If the Eluate Discard step is selected, the specified volume is dispensed into the Organic Waste plate or Flow Through Collection plate (if the Add to Flow Through step is selected). The remaining Elution Buffer is dispensed through cartridges at the Elute flow rate 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 = 15 µL (5 µL cartridges) or 40 µL (25 µL cartridges) Eluate Discard volume = 2 µL (5 µL cartridges) or 10 µL (25 µL cartridges) the first 2 µL (5 µL cartridges) or 10 µL (25 µL cartridges) eluate from the cartridges will be discarded into the Flow Through Collection plate, and the remaining 13 µL (5 µL cartridges) or 30 µL (25 µL cartridges) eluate will be collected in the Eluate Collection plate. Select the Eluate Discard step in situations where minimizing the volume of eluate is important. For AssayMAP cartridges, the initial elution volume (~2 µL for 5 µL cartridges or ~10 µL for the 25 µL cartridges) contains small or no measurable amounts of analyte. This step 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: 0–2 – 25 µL cartridges: 0–10 • Range: 0–250 |
Add to Flow Through | If selected, this step dispenses the Eluate Discard volume into the Flow Through Collection plate. If the Add to Flow Through step is not selected, the Eluate Discard is dispensed into the Organic Waste plate. This step is not selected by default. Note: The Add to Flow Through step is an option only if both the Elute and Eluate Discard steps have been selected. |
Existing Collection Volume | This step enables you to specify an amount of liquid that is in the wells of the Eluate Collection plate at the beginning of the run. 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 the labware for the Eluate Collection plate, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Select this step when the Eluate Collection plate contains a volume of liquid useful for immediately diluting the eluates, for adjusting the pH of the eluates, or to aid in the recovery of small volumes of eluates from AssayMAP cartridges. Volume (µL): • Default: 0 • Practical: 0—250 • Range: 0—300 |
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 of DI water 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: 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
This section describes the basic movements of the AssayMAP Bravo Platform during the Peptide Cleanup protocol using the default method. Changing the selections or parameters will alter the movements.
Protocol step | Head moves to deck location... | Action |
|---|---|---|
Starting protocol | 2 | Parks all cartridges that might have been loaded 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. |
Prime | 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 exterior of the syringe probes. | |
5 | 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. | |
5 | Aspirates the Priming Buffer. | |
2 | Mounts the cartridges onto the head. | |
3 | Dispenses the Priming Buffer through the cartridges and into Organic Waste plate to prime the cartridges. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
Equilibrate | 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 exterior of the syringe probes. | |
8 | 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. | |
8 | Aspirates the Equilibration Buffer. | |
2 | Mounts the cartridges on the head. | |
3 | Dispenses the Equilibration Buffer through the cartridges to equilibrate. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
Load Samples | 4 | Aspirates the samples into the syringes. |
1 | Washes the exterior of the syringe probes. | |
2 | Mounts the cartridges on the head. | |
7 | 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 | 8 | Aspirates the Cartridge Wash Buffer. |
2 | Washes the cartridge cups and exercises the cartridges off task. | |
3 | Dispenses the Cartridge Wash Buffer into the Organic Waste plate. | |
1 | Washes the syringes. | |
Internal Cartridge 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 exterior of the syringe probes. | |
8 | Aspirates 10 µL of Cartridge Wash Buffer for the cartridge air-gap prevention step. | |
2 | Dispenses the remaining 10 µL of buffer into the cartridge cups and exercises the cartridges off task. | |
8 | Aspirates the Cartridge Wash Buffer into the syringes for sample chase and the Internal Cartridge Wash steps. | |
2 | Mounts the cartridges on the head. | |
3 | Dispenses 5 µL (5 µL cartridges) or 25 µL (25 µL cartridges) Cartridge Wash Buffer through the cartridges at the Load Samples flow rate for the sample chase step. | |
3 | Dispenses the remaining Cartridge 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. | |
Stringent Syringe Wash | 5 | Aspirates the Syringe Wash Buffer. |
3 | Dispenses the Syringe Wash Buffer into the Organic Waste plate. | |
1 | Washes the syringes. | |
Elute | 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 exterior of the syringe probes. | |
6 | Aspirates 10 µL of Elution Buffer for the cartridge air-gap-prevention step. | |
2 | Dispenses the 10 µL of Elution Buffer into the cartridge cups and exercises the cartridges off task. | |
6 | Aspirates the Elution Buffer. | |
2 | Mounts the cartridges on the head. | |
9 | Elutes the samples into the Eluate Collection plate. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
9 | Mixes the eluates. | |
1 | Washes the syringes. | |
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. |