Assay development guidelines and protocol notes

This topic explains each step of the protocol so that you can optimize the Phosphopeptide Enrichment protocol to your particular experimental design.

Note: For guidelines on performing a mock run, see About performing a mock run (optional).

Protocol stepwise guidelines

 

Cartridges ship dry and, therefore, contain air entrained in the cartridge bed. Failure to prime the cartridges can prevent the sample and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

 

The first steps in the Phosphopeptide Enrichment protocol set up the system and prepare the cartridges for sample loading, including:

•	Washing the syringes in the Bravo 96AM head to ensure there is no carryover from previous experiments.

•	Priming the cartridges to condition the resin and remove entrained air.

•	Equilibrating the cartridges to create optimal conditions for binding.

 

Do not allow wetted cartridges to dry out.

 

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 cause damage to both the cartridges and the AssayMAP syringes in the head.

For example, if the software specifies 10 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 do to liquid moving past the syringe cartridges seal into the cartridge cup.

Default: 1

Range: 1-12

Initial Syringe Wash step

The Initial Syringe Wash step removes any potential contaminants from the syringes. 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.

Select the Initial Syringe Wash step to clean the syringes. Skip this step if you are certain that the syringes are clean. This step is selected by default.

Table Setting the Initial Syringe Wash parameter

 

Parameter	Value	Notes
Wash Cycles	Default: 3 Practical: 3–5 Range: 0–10	You can increase the number of wash cycles to better clean the syringes. However, increasing the number of wash cycles beyond the practical range serves only to increase the time of the assay, unnecessarily consume reagents, and cause additional wear on the syringes.

Prime step

The Prime step removes entrained air from within the cartridges and properly wets the surface of the resin. For the most effective priming of AssayMAP Fe(III)-NTA and TiO2 cartridges, the Priming Buffer requires that the solution contain at least 50% organic solvent. Higher concentrations of organic solvent are also acceptable.

The Prime step aspirates the specified volume of Priming Buffer (deck location 5) into the syringes, mounts the cartridges (deck location 2) onto the syringes, and then dispenses buffer at the specified flow rate through the cartridges into the Organic Waste plate (deck location 3). After removing the cartridges at the seating station (deck location 2), the syringes are washed the specified number of cycles at the wash station (deck location 1) using 250 µL per wash cycle.

Note: The Priming Buffer (deck location 5) is also used for the Syringe Wash Buffer.

Always perform the Prime step at the beginning of a run. In the default protocol, this step is selected.

Table Setting the Prime step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 100 Practical: 100–200 Range: 0–250	The default volume is equal to 20 column volumes, which should be sufficient to wet the cartridge resin and remove entrained air from within the cartridge. Note: Setting the Volume at 0 skips all Prime step tasks, except syringe washing.
Flow Rate (µL/min)	Default: 300 Practical: 250–350 Range: 0.5–500	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 300 µL/min is not required and has not been tested.
Wash Cycles	Default: 1 Practical: 1–3 Range: 0–10	This parameter specifies the number of syringe wash cycles performed at the wash station after the step. Each wash cycle aspirates 250 µL liquid from the chimneys, and then dispenses the liquid to waste.

Equilibrate step

The Equilibrate step exchanges the Priming Buffer with Equilibration Buffer within the resin bed. Equilibrating the cartridge bed with a buffer that establishes optimal chemical conditions for sample binding is critical for the Load Samples step.

The Equilibrate step aspirates the specified volume of Equilibration Buffer (deck location 8) into the syringes, mounts the cartridges (deck location 2) onto the syringes, and then dispenses the buffer at the specified flow rate through the cartridges into the Organic Waste plate (deck location 3). After removing the cartridges at the seating station (deck location 2), the syringes are washed the specified number of cycles at the wash station (deck location 1) using 250 µL per wash cycle.

Note: The Equilibration Buffer (deck location 8) is also used for the Cartridge Wash Buffer.

Always perform the Equilibrate step after cartridge priming to establish optimum sample loading conditions. In the default protocol, this step is selected.

Table Setting the Equilibrate step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 50 Practical: 50–100 Range: 0–250	The default volume, 50 µL, is equal to 10-column volumes, which should be sufficient for complete buffer exchange. Note: Setting the Volume at 0 skips all Equilibrate step tasks, except syringe washing.
Flow Rate (µL/min)	Default: 10 Practical: 5–25 Range: 0.5–500	A flow rate < 10 µL/min will likely have no benefit, but will increase the total assay time. A flow rate > 25 µL/min using the default volume might not equilibrate through the pores in the beads across the full length of the cartridge bed.
Wash Cycles	Default: 1 Practical: 1–3 Range: 0–10	This parameter specifies the number of syringe wash cycles performed at the wash station after the step. Each wash cycle aspirates 250 µL liquid from the chimneys, and then dispenses the liquid to waste.

Load Samples step

The Load Samples step aspirates the samples (deck location 4) into the syringes, and then performs an external syringe probe wash at the wash station to remove any sample remaining on the outside of the probes before mounting the cartridges at the seating station (deck location 2). The sample is dispensed through cartridges at the specified flow rate into the Flow Through Collection plate (deck location 7) if the Collect Flow Through step is selected, or into the Organic Waste plate (deck location 3). During an external cartridge tip wash, the cartridge tips dip into the wash station chimneys (deck location 1) to remove any sample on the outside of the cartridge. After removing the cartridges at the seating station (deck location 2), the syringes are washed the specified number of cycles at the wash station (deck location 1) using 250 µL per wash cycle.

The Phosphopeptide Enrichment protocol permits up to 1000 µL of sample to be dispensed through the cartridges. However, each syringe has a maximum capacity of 250 µL. For sample volumes >250 µL, the protocol will iteratively load equal volumes onto the cartridges during the Load Samples step.

For example, to load 280 µL of sample onto cartridges from U-bottom plates, the protocol will load the sample in two 140 µL loading loops. Similarly, to load 900 µL from a deep-well plate, the protocol will load the sample in four sample loading loops of 225 µL each.

The protocol uses the following formula to determine the number and volume of iterative load steps:

•	# 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

The protocol uses logic to limit the amount of sample that can be aspirated from specific labware types. For example, an error message will appear at the beginning of a run if the volume of the Load Samples step is 500 µL and the labware selection for the Samples plate at deck location 4 is a PCR plate. This logic minimizes the likelihood of overflowing samples into adjacent wells when the probes are immersed in the wells to aspirate sample. This logic also adjusts the well-bottom offset and facilitates sample volume-dependent head movement based on the volume in the Load Samples step.

For the maximum practical working volumes per well for specific labware at deck location 4, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Always select the Load Samples step during a normal run. In the default protocol, this step is selected.

Table Setting the Load Samples step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 100 Practical: 25–250 Range: 0–1000	The volume of sample should be balanced with the sample concentration and the mass capacity of the cartridge. It may not be possible to aspirate all of the liquid from a microplate well reproducibly. Add at least 10‑ to 35-µL extra sample to each well (labware dependent) to aspirate reliably the desired volume of sample. This also ensures that the sample does not deplete fully, which would result in the aspiration of air into the syringe and then into the cartridges, compromising performance. Large sample volumes (> 250 µL) may require slightly more excess sample due to evaporation. Note: Setting the Volume at 0 skips all the Load Samples step tasks, except syringe washing.
Flow Rate (µL/min)	Default: 5 Practical: 2–15 Range: 0.5–500	The optimum sample loading flow rate requires balancing the speed of the assay and desired recovery. The binding capacity is inversely proportional to flow rate. For some samples, a flow rate > 15 µL/min can result in a low dynamic binding capacity. It may 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. Flow rates < 2 µL/min may not provide significant increases in analyte binding.
Wash Cycles	Default: 3 Practical: 2–5 Range: 0–10	This parameter specifies the number of syringe wash cycles performed at the wash station after the step. Each wash cycle aspirates 250 µL liquid from the chimneys, and then dispenses the liquid to waste.

Collect Flow Through (from the Load Samples step)

Note: The Collect Flow Through step is an option only if the Load Samples step is conducted.

If you select the Collect Flow Through step, the sample flow-through from the Load Samples step is dispensed directly into the Flow Through Collection plate (deck location 7).

If the Load Samples step is conducted, but the Collect Flow Through step is not selected, the sample flow-through will be dispensed directly into the Organic Waste plate (deck location 3).

For the maximum practical working volumes per well for specific labware at deck location 7, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Select the Collect Flow Through step if you want to collect the sample flow-through generated during the Load Samples step for downstream processing or for analysis.

Cup Wash step

The Cup Wash step removes the small volume of residual sample liquid that might remain above the upper cartridge membrane after sample loading. This step effectively rinses the cup portion of AssayMAP cartridges with buffer. The Cartridge Wash Buffer (deck location 8) is aspirated into the syringes, and then dispensed into the cartridge cups (deck location 2). This liquid, plus any residual liquid from samples, is aspirated from the cartridge cups into the syringes. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the Organic Waste plate (deck location 3), and then washing the syringes at the wash station (deck location 1).

Note: The Cartridge Wash Buffer (deck location 8) is also used for the Equilibration Buffer.

Select the Cup Wash step if there is residual sample liquid in the cartridge cups that might cause complications with analysis.

Table Setting the Cup Wash step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 25 Practical: 25–50 Range: 0–100	Specify the volume of Cartridge Wash Buffer to dispense into the cartridge cup. A volume < 10 µL may be insufficient for cup washing. A volume > 50 µL may offer little benefit. Note: Setting the Volume at 0 skips all tasks associated with the Cup Wash step.
Wash Cycles	Default: 1 Practical: 1–3 Range: 1–10	A wash cycle is defined as a cup wash, plus a syringe wash with 250 µL of liquid from the wash station. Use the Wash Cycles setting to control the number of times this step repeats.

Internal Cartridge Wash step

The Internal Cartridge Wash step aspirates Cartridge Wash Buffer (deck location 8) into the syringes. After mounting the cartridges at the seating station (deck location 2), the buffer is dispensed through the cartridges at the specified flow rate into the Flow Through Collection plate (deck location 7), if the Collect Flow Through step is selected, or into the Organic Waste plate (deck location 3). An external cartridge tip wash is performed at the wash station (deck location 1), where the cartridge tips dip into the chimneys to remove any sample on the outside of the cartridge. After removing the cartridges at the seating station (deck location 2), the syringes are washed the specified number of cycles at the wash station (deck location 1) using 250 µL per wash cycle.

Note: The Cartridge Wash Buffer (deck location 8) is also used for the Equilibration Buffer.

If the Load Samples step was selected, an additional 5 µL of Cartridge Wash Buffer is aspirated for the sample chase in addition to the specified volume for the Internal Cartridge Wash step. The sample chase uses the Load Samples flow rate to dispense buffer through the cartridges so that all sample loaded onto the cartridge has an opportunity to interact with the entire bed of the cartridge (1 bed volume = 5 µL). After the first 5 µL of buffer has been dispensed through the cartridge, the remaining buffer is dispensed through the cartridges at the Internal Cartridge Wash flow rate. If the Load Samples step is skipped, the sample chase step is also skipped.

Select the Internal Cartridge Wash step to minimize non-target elements from the sample matrix in the cartridge resin bed, which could possibly contaminate the eluate. In the default protocol, this step is selected.

Table Setting the Internal Cartridge Wash step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 50 Practical: 50–100 Range: 0–250	This parameter specifies the volume of Cartridge Wash Buffer to dispense through cartridges to wash the resin bed. Note: Setting the Volume at 0 skips all tasks associated with the Internal Cartridge Wash step, except syringe washing.
Flow Rate (µL/min)	Default: 10 Practical: 5–25 Range: 0.5–500	A flow rate < 10 µL/min will likely have little benefit, but will increase the total assay time. A flow rate > 25 µL/min might not equilibrate through the pores in the resin across the full length of the cartridge bed, which can result in incomplete washing.
Wash Cycles	Default: 3 Practical: 2–5 Range: 0–10	This parameter specifies the number of syringe wash cycles performed at the wash station after the step. Each wash cycle aspirates 250 µL liquid from the chimneys, and then dispenses the liquid to waste.

Collect Flow Through step (from the Internal Cartridge Wash step)

Note: The Collect Flow Through step is an option only if the Internal Cartridge Wash step is conducted.

The Collect Flow Through step enables the wash flow-through from the Internal Cartridge Wash step to be directly eluted into the Flow Through Collection plate (deck location 7) during the Internal Cartridge Wash step. If the Internal Cartridge Wash step is conducted, but the Collect Flow Through step is not selected, the wash flow-through is dispensed directly into the Organic Waste plate (deck location 3).

For the maximum practical working volumes per well for specific labware at deck location 7, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Select this step if you want to collect the flow-through generated during the Internal Cartridge Wash step and combine it with the flow-through generated during the Load Samples step for downstream processing or analysis. In the default protocol, this step is not selected.

Stringent Syringe Wash step

The Stringent Syringe Wash step comprehensively cleans the syringes with Syringe Wash Buffer (deck location 5) to reduce the potential for sample carryover. The step aspirates buffer into the syringes (deck location 5), moves the head moves above the Organic Waste plate (deck location 3), draws the buffer up to the maximum available volume of the syringe, and then moves the head down and dispenses the buffer into the Organic Waste plate. The head then moves to the wash station (deck location 1) and washes the syringes with 250 µL of liquid per wash cycle.

Note: The Syringe Wash Buffer (deck location 5) is also used for the Priming Buffer.

Select the Stringent Syringe Wash step when rigorous cleaning of the syringes is necessary to prevent carryover. In the default protocol, this step is selected.

Table Setting the Stringent Syringe Wash step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 50 Practical: 50–100 Range: 0–250	This parameter specifies the volume of Syringe Wash Buffer to aspirate into the syringes and dispense into the Organic Waste plate to stringently wash syringes. Note: Setting the Volume at 0 skips all tasks associated with the Stringent Syringe Wash step.
Wash Cycles	Default: 1 Practical: 1–3 Range: 1–10	A cycle is defined as a stringent syringe wash, plus a syringe wash at the wash station. Use the Wash Cycles setting to control the number of times this step repeats.

Elute step

The Elute step uses Elution Buffer to elute bound analytes from AssayMAP cartridges. The Elute step aspirates the specified volume of Elution Buffer into syringes (deck location 6), mounts the cartridges at the seating station (deck location 2), and then dispenses the Elution Buffer through the cartridges at the specified flow rate into the Eluate Collection plate (deck location 9). An external cartridge tip wash is performed at the wash station (deck location 1), where the cartridge tips dip into the chimneys to remove any eluate on the outside of the cartridge. After removing the cartridges at the seating station (deck location 2), the head moves back to deck location 9 where eluates are mixed in the Eluate Collection plate. (If the total volume in the Eluate Collection plate is < 15 µL, the eluates will not be mixed). The head then moves to the wash station (deck location 1) and washes the syringes for the specified number of wash cycles using 250 µL per wash cycle.

Using sufficiently strong elution conditions and the default flow rate (5 µL/min), phosphopeptides can be fully eluted from cartridges in as little as 10 µL (approximately 2 cartridge column volumes).

The maximum volume of eluate that can be collected in the Eluate Collection plate (deck location 9) is dependent on the maximum practical working volume of the labware defined for the Phosphopeptide Enrichment protocol. The total volume in the Eluate Collection plate is determined as follows:

Total volume = Net Elution Volume (Elute volume - Eluate Discard volume) + Existing Collection Volume

For the maximum practical working volumes per well for specific labware at deck location 9, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Always select the Elute step to elute bound analytes from cartridges in a normal run. In the default protocol, this step is selected.

Table Setting the Elute step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 20 Practical: 10–30 Range: 0–250	This parameter specifies the volume of Elution Buffer aspirated into the syringes from deck location 6 and dispensed through the cartridges into the Eluate Collection plate at deck location 9. Note: Setting the Volume at 0 skips all tasks associated with the Elute step, except syringe washing.
Flow Rate (µL/min)	Default: 5 Practical: 2–15 Range: 0.5–500	A flow rate < 2 µL/min is unlikely to improve the elution yield. Elution yield can be compromised if the flow rate is > 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).
Wash Cycles	Default: 1 Practical: 1–3 Range: 0–10	This parameter specifies the number of syringe wash cycles performed at the wash station after the step. Each wash cycle aspirates 250 µL liquid from the chimneys, and then dispenses the liquid to waste.

Eluate Discard step

Note: The Eluate Discard step is an option only if the Elute step is conducted.

The Eluate Discard step permits a specified volume of the first eluate from the AssayMAP cartridges to be discarded. The Eluate Discard step aspirates the specified volume of Elution Buffer (deck location 6) from the Elute step into the syringes, mounts the cartridges at the seating station (deck location 2), and dispenses the Elution Buffer through the cartridges at the specified Elute flow rate into the Organic Waste plate (deck location 3) or the Flow Through Collection plate (deck location 7), 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 (deck location 9).

For example, if you select the Elute, Eluate Discard, and Add to Flow Through steps and set the Elute volume = 20 µL and the Eluate Discard volume = 3 µL, the first 3 µL of eluate from the cartridges will be discarded to the Flow Through Collection plate (deck location 7) and the next 17 µL of eluate will be collected in the Eluate Collection plate (deck location 9).

For some experimental situations, minimizing the total volume of sample eluates is critical to downstream processing. This may be the case when the concentration of organic solvent must be diluted to suitable levels for reversed-phase LC/MS analyses, but the total sample volume must stay within the allowable injection volume in order to inject the entire sample. In this case, it can be valuable to discard the first few microliters of eluate from an AssayMAP cartridge, which do not contain measurable amounts of analyte.

For AssayMAP cartridges, the first 2 to 5 µL of eluate contains small or no measurable amounts of analyte. The volume of the analyte-free eluate will vary depending on both the strength of the elution buffer and the degree of analyte saturation of the cartridges. This value should be determined empirically for each combination of sample, buffer, and cartridge type for a given experiment.

Select the Eluate Discard step if minimizing the volume of eluate is critical. In the default protocol, this step is not selected.

Table Setting the Eluate Discard step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 0 Practical: 2–5 Range: 0–25	This parameter specifies the first volume of eluate that will be discarded during the Elute step. The value can equal, but cannot exceed the Elute volume.
Flow Rate (µL/min)	Same as the Elute step	See the Elute step.

Add to Flow Through step

Note: The Add to Flow Through step is an option only if both the Elute and Eluate Discard steps have been selected.

If selected, the Add to Flow Through step dispenses the Eluate Discard volume into the Flow Through Collection plate (deck location 7). If the Add to Flow Through step is not selected, the Eluate Discard is dispensed into the Organic Waste plate (deck location 3).

For the maximum practical working volumes per well for specific labware at deck location 7, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Select the Add to Flow Through step if both the Elute and Eluate Discard steps have been selected and the Eluate Discard volume is to be added to the Flow Through Collection plate at deck location 7. In the default protocol, this step is not selected.

Existing Collection Volume step

This step enables users to specify an amount of liquid that is already contained in the wells of the Eluate Collection plate (deck location 9). The protocol uses the Existing Collection Volume and the net volume from the Elute step (Elute Volume - Eluate Discard Volume) to adjust the well-bottom offset for sample elution, calculate the eluate mixing volume, and dynamically move the head in and out of the wells during elution and eluate mixing in a volume-dependent manner.

For the maximum practical working volumes per well for specific labware at deck location 9, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Select the Existing Collection Volume step if the Eluate Collection plate contains some volume of liquid useful for immediately diluting the eluates, for immediately adjusting the pH of the eluates, or to aid in the recovery of small volumes of eluates from the AssayMAP cartridges.

Table Setting the Existing Collection Volume step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 0 Practical: 0–50 Range: 0–300	This parameter specifies the volume of liquid in the wells of the Eluate Collection plate at deck location 9 before elution.

Final Syringe Wash step

The Final Syringe Wash step uses the wash station (deck location 1) to flush any potential contaminants from the syringes in the head. 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.

Select the Final Syringe Wash step after each run to clean the syringes thoroughly before running a different assay. In the default protocol, this step is selected.

Table Setting the Final Syringe Wash parameter

 

Parameter	Value	Notes
Wash Cycles	Default: 3 Practical: 3–5 Range: 0–10	This parameter specifies the number of final syringe washes upon completion of the protocol. Increase the number of wash cycles to better clean the syringes.

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.

 

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Automation movements during the protocol

This section describes the basic automation movements of the AssayMAP Bravo Platform during the Phosphopeptide Enrichment protocol when the protocol defaults are used. Changing the selections or deselecting options will alter the movements. The default protocol takes approximately 50 minutes to complete.

 

Protocol step	Head moves to deck location...	Action
Run Cartridge Transfer utility	6	Mounts one column of Fe(III)-NTA or TiO2 cartridges from column A of the 96AM Cartridge Rack and Receiving Plate.
	2	Parks the cartridges in column A of the 96AM Cartridge & Tip Seating Station.
Starting protocol	2	Checks the head and parks all cartridges that might have been loaded on the head from a previous protocol.
	1	Dispenses any liquid remaining in the syringes into the wash station.
Initial Syringe Wash	1	Washes the syringes for 3 cycles.
Prime	5	Aspirates the Priming Buffer into the syringes.
	2	Mounts the cartridges onto the head.
	3	Dispenses the Priming Buffer through the cartridges into the Organic Waste plate to prime the cartridges.
	2	Parks the cartridges.
	1	Washes the syringes for 1 cycle.
Equilibrate	8	Aspirates the Equilibration Buffer into the syringes.
	2	Mounts the cartridges on the head.
	3	Dispenses the Equilibration Buffer through the cartridges into the Organic Waste plate to equilibrate the cartridges.
	2	Parks the cartridges.
	1	Washes the syringes for 1 cycle.
Load Samples	4	Aspirates the samples into the syringes.
	1	Washes exterior of the syringe probes at the wash station.
	2	Mounts the cartridges on the head.
	7	Dispenses the samples through the cartridges into the Flow Through Collection plate.
	1	Washes the exterior of the cartridges at the wash station.
	2	Parks the cartridges.
	1	Washes the syringes for 3 cycles.
Cup Wash	8	Aspirates the Cartridge Wash Buffer into the syringes.
	2	Washes the cartridge cups.
	3	Dispenses the Cartridge Wash buffer into the Organic Waste plate.
	1	Washes the syringes for 1 cycle.
Internal Cartridge Wash	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 of the Cartridge Wash Buffer through the cartridges into the Organic Waste plate at the Load Samples flow rate for the sample chase.
	3	Dispenses the Cartridge Wash buffer through the cartridges into the Organic Waste plate.
	1	Washes the exterior of the cartridge tips at the wash station.
	2	Parks the cartridges.
	1	Washes the syringes for 3 cycles.
Stringent Syringe Wash	5	Aspirates the Syringe Wash Buffer into the syringes.
	3	Dispenses the Syringe Wash Buffer into the Organic Waste plate.
	1	Washes the syringes for 1 cycle.
Elute	6	Aspirates the Elution Buffer into the syringes.
	2	Mounts the cartridges on the head.
	9	Elutes the samples into the Elution Collection Plate.
	1	Washes the exterior of the cartridge tips at the wash station.
	2	Parks the cartridges.
	9	Mixes the eluates.
	1	Washes the syringes for 1 cycle.
Final Syringe Wash	1	Washes the syringes for 3 cycles.

 

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