Assay development guidelines and protocol notes

This topic explains each step of the Protein Cleanup v2.0 protocol so that you can optimize the protocol to your particular experimental design.

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 removes 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 wash station chimneys to dispense to waste. You can increase the number of wash cycles to better clean the syringes. However, more cycles increases the total run time and causes wear on the syringes. This step is selected by default. Wash Cycles: • Default: 3 • Practical: 3–5 • Range: 0–10
Prime	This step removes entrained air from within the cartridges and properly wets the surface of the resin. Priming of AssayMAP RP‑W cartridges requires a Priming & Syringe Wash Buffer that contains at least 25% organic solvent. Higher concentrations of organic solvent are also acceptable. Before the priming begins, 10 µL of Priming & Syringe Wash Buffer is aspirated 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 specified volume of buffer into the syringes, mounts the cartridges on the syringes, and then dispenses buffer at the specified flow rate through the cartridges into the Organic Waste plate. After parking the cartridges at the seating station, the syringes are washed the specified number of cycles at the wash station using 250 µL per wash cycle. Always perform the Prime step at the beginning of a run. This step is selected by default. Volume (µL). 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. 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 – Range: 0–250 • Volume for 25 µL cartridge: – Default: 250 – Range: 0–250 Note: Setting the volume to zero will skip all tasks associated with the Prime step except syringe washing at the wash station. Flow rate (µL/min). A flow rate slower 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: 250–350 • Range: 0.5–500 Wash cycles. 250 µL of DI water is used for each syringe wash cycle. • Default: 1 • Practical: 1–3 • Range: 0–10
Equilibrate	This step exchanges the Priming & Syringe Wash Buffer with Equilibration & Cartridge Wash Buffer within the resin bed. Equilibrating the cartridge bed with a buffer that establishes optimal chemical conditions for sample binding. The Equilibrate step aspirates the specified volume of Equilibration & Cartridge Wash Buffer into the syringes, mounts the cartridges onto the syringes, and then dispenses the buffer at the specified flow rate through the cartridges into the Organic Waste plate. After removing the cartridges at the seating station, the syringes are washed the specified number of cycles at the wash station using 250 µL per wash cycle. Always perform the Equilibrate step after cartridge priming to establish optimum sample loading conditions. This step is selected by default. Volume (µL). The default volume is equal to 10 column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. • Volume for 5 µL cartridge: – Default: 50 – Range: 0–250 • Volume for 25 µL cartridge: – Default: 250 – Range: 0–250 Note: Setting the volume to zero will skip all tasks associated with the Equilibrate step except syringe washing at the wash station. 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 might not equilibrate through the pores in the beads. • Default: 10 • Practical: 5–25 • Range: 0.5–500 Wash cycles. 250 µL of DI water is used for each syringe wash cycle. • Default: 1 • Practical: 1–3 • Range: 0–10
Load Samples	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 at the seating station. The sample is dispensed through cartridges at the specified flow rate into the Flow Through Collection plate if the Collect Flow Through step is selected, or into the Organic Waste plate. During an external cartridge tip wash, the cartridge tips dip into the wash station chimneys to remove any sample on the outside of the cartridge. After removing the cartridges at the seating station, the syringes are washed the specified number of cycles at the wash station using 250 µL per wash cycle. The protocol accommodates sample volumes up to 1000 µL to be dispensed 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. For sample volumes greater than 250 µL, the protocol will iteratively load equal volumes onto the cartridges. To determine the number and volume of iterative load steps, 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 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. IMPORTANT The volume of sample to load should be balanced with the sample concentration and the mass capacity of the cartridge. Be sure to include the recommended labware-specific volume overage to prevent air from entering the cartridge. For more information, see Transferring the samples to the microplate. 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 • Range: 0–1000 Note: Setting the volume to zero skips all Load Samples tasks except for syringe washing. Flow rate (µL/min). The optimum sample loading flow rate requires balancing the speed of the assay and desired recovery. Binding capacity is inversely proportional to the flow rate. For some samples, a flow rate faster than 15 µL/min can result in a low dynamic binding capacity. It might also produce some loss of total binding capacity, as the sample does not have sufficient time to diffuse throughout the pores of the cartridge resin bed. Using flow rates slower than 5 µL/min might not significantly increase analyte binding. • Default: 5 • Practical: 2–15 • Range: 0.1–500 Wash cycles. 250 µL of DI water is used for each syringe wash cycle. • Default: 3 • Practical: 2–5 • Range: 0–10
Collect Flow Through	If 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 sample flow-through is dispensed in the Organic Waste plate. This step is selected by default.
Cup Wash	This step removes the small volume of residual sample liquid that might remain above the upper cartridge membrane after sample loading. The Cartridge Wash Buffer is aspirated into the syringes, and then dispensed into the cartridge cups. 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. Volume (µL). A volume <10 µL might 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 will skip all tasks associated with the Cup Wash step. Wash cycle. Each cycle comprises one cup wash and one syringe wash. • Default: 2 • Practical: 1–3 • Range: 1–10 Note: Each wash cycle consists of a cup wash followed by syringe wash.
Internal Cartridge Wash	Uses Equilibration & Cartridge Wash Buffer to wash non-specifically bound molecules from the cartridge resin. Before the cartridge wash begins, 10 µL of Equilibration & Cartridge Wash Buffer is aspirated into the syringes and dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Internal Cartridge Wash step aspirates buffer into the syringes, mounts the cartridges on the syringes, and then dispenses the buffer through the cartridges at the specified flow rate into the Flow Through Collection plate, if the Collect Flow Through step is selected, or into the Organic Waste plate. An external cartridge tip wash is performed at the wash station, 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, the syringes are washed the specified number of cycles at the wash station using 250 µL per wash cycle. If the Load Samples step was selected, an additional 25 µL of Cartridge Wash Buffer is aspirated in addition to the specified volume for the Internal Cartridge Wash step. This additional volume is the sample chase and it is dispensed at the Load Samples flow rate before the Internal Cartridge Wash begins. 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. If the wash volume plus the sample chase equal >250 µL, the sample chase is performed with part of the wash volume. This step is selected by default. Volume (µL). The volume to dispense through the cartridges to wash the resin bed. Volumes higher than the default volume may improve the purification marginally but also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. • Volume for 5 µL cartridges: – Default: 50 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 250 – Range: 0–250 Flow rate (µL/min). A rate slower than the default flow rate will have little benefit, but will increase the total assay time. A rate faster than 20 µL/min might not equilibrate through the pores in the beads, resulting in incomplete washing. • Default: 10 • Practical: 5–20 • Range: 0.5–500 Wash cycle. The number of syringe wash cycles to perform at the end of the task. 250 µL of DI water is used for each syringe wash cycle. • Default: 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 in the Flow Through Collection plate. If the Collect Flow Through step is not selected, the wash flow-through is dispensed in the Organic Waste plate. This step is not selected by default. Note: The Collect Flow Through step is an option only if the Internal Cartridge Wash step is conducted.
Stringent Syringe Wash	This step cleans the syringes with Priming & Syringe Wash Buffer prior to elution. This step aspirates the Syringe Wash Buffer into the syringes, moves the head above the Organic Waste plate, 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 syringes are washed at the wash station with 250 µL of liquid per wash cycle. This step is selected by default. Volume (µL). The volume of buffer to aspirate and dispense. Volumes higher than the default volume are unlikely to improve the syringe cleaning but will increase the run time and reagent consumption. Volumes lower than the default volume may be insufficient for syringe washing. • Default: 50 • Range: 0–250 Note: Setting the volume to zero will skip all tasks associated with the Stringent Syringe Wash step. Wash cycle. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. • Default: 2 • Practical: 1–3 • Range: 1–10
Elute	This step uses Elution Buffer to elute bound proteins from the cartridges. The Elute step aspirates the specified volume of Elution Buffer into syringes, mounts the cartridges at the seating station, and then dispenses the buffer through the cartridges at the specified flow rate 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. The syringes are washed at the wash station for the specified number of wash cycles, using 250 µL per cycle. 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. After all the Elute operations, this step aspirates any excess liquid remaining in the cartridge cups, dispenses the liquid at the wash station, and then performs a final syringe wash. This step is selected by default. Volume (µL): Using sufficiently strong elution conditions and the default flow rate (5 µL/min), proteins can be fully eluted from cartridges in approximately 2 to 3 cartridge column volumes. The maximum volume of eluate that can be collected in the Eluate Collection plate is dependent on the maximum practical working volume of the labware used. 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, 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 will skip all tasks associated with the Elute step except syringe washing at the wash station.
	Flow rate (µL/min). A flow rate slower than 5 µL/min is unlikely to improve the elution yield. Elution yield may be compromised if flow rates are faster than 15 µL/min for a given volume of elution buffer (that is, more elution buffer may be required to get the same elution yield at high elution flow rates relative to using lower flow rates for a given elution volume). • Default: 5 • Practical: 2–15 • Range: 0.1–500 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
Eluate Discard	This step permits a specified volume of the first eluate from the cartridges to be discarded. If selected, the Eluate Discard step dispenses the specified volume into the Organic Waste plate, or into the 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: 25 µL • Eluate Discard volume: 5 µL the first 5 µL eluate from the cartridges will be discarded in the Flow Through Collection plate, and the remaining 20 µL eluate will be collected in the Eluate Collection plate. Select the Eluate Discard step in situations were minimizing the volume of the eluate is critical. 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 initial elution volume (~2 µL for 5 µL cartridges and ~10 µL for the 25 µL cartridges) 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. This step is not selected by default. Note: The Eluate Discard step is an option only if the Elute step is conducted. 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: 2 – 25 µL cartridges: 10 • Range: 0–250
Add to Flow Through	If selected, the Add to Flow Through 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 users to specify an amount of liquid that is already contained in the wells of the Eluate Collection plate. 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 in and out of the wells during elution and eluate mixing in a volume-dependent manner. Select the Existing Collection Volume step if the Eluate Collection plate contains some 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 the AssayMAP cartridges. Volume (µL) • Default: 0 • Range: 0–300
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. Note: The amount of carryover in the syringe (expressed as dilution of the starting sample) depends on the total number of syringe wash cycles. 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. Select the Final Syringe Wash step after each run to clean the syringes thoroughly. This step is selected by default. Wash Cycles: • Default: 3 • Practical: 3–5 • Range: 0–10

 

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Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Protein Cleanup protocol. 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	5	Aspirates 10 µL of Priming & Syringe Wash 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 & Syringe Wash Buffer.
	2	Mounts the cartridges onto the head.
	3	Dispenses the buffer through the cartridges into the Organic Waste plate to prime the cartridges.
	2	Parks the cartridges.
	1	Washes the syringes.
Equilibrate	8	Aspirates the Equilibration & Cartridge Wash Buffer.
	2	Mounts the cartridges on the head.
	3	Dispenses the buffer through the cartridges to equilibrate.
	2	Parks the cartridges.
	1	Washes the syringes.
Load Samples	4	Aspirates the samples into the syringes.
	1	Washes syringe exterior.
	2	Mounts the cartridges on the head.
	7	Dispenses the samples through the cartridges.
	1	Washes the cartridge exteriors.
	2	Parks the cartridges.
	1	Washes the syringes.
Cup Wash	8	Aspirates the Equilibration & Cartridge Wash Buffer.
	2	Washes the cartridge cups and exercises the cartridges off task.
	3	Dispenses the buffer.
	1	Washes the syringes.
Internal Cartridge Wash	8	Aspirates 10 µL of Equilibration & Cartridge Wash 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.
	8	Aspirates the Cartridge Wash Buffer into the syringes for the sample chase and the Internal Cartridge Wash steps.
	2	Mounts the cartridges on the head.
	3	Dispenses the sample chase through the cartridges at the Load Samples flow rate.
	3	Dispenses the Cartridge Wash buffer through the cartridges.
	2	Parks the cartridges.
	1	Washes the syringes.
Stringent Syringe Wash	5	Aspirates the Priming & Syringe Wash Buffer.
	3	Dispenses the buffer.
	1	Washes the syringes.
Elute	6	Aspirates the Elution Buffer.
	2	Mounts the cartridges on the head.
	9	Elutes the samples into the Elution Collection plate.
	1	Washes the cartridge exteriors.
	2	Parks the cartridges.
	9	Mixes the eluates.
	1	Washes the syringes.
	2	Aspirates excess liquid from the cartridge cups, and exercises the cartridges off task.
	1	Dispenses liquid to waste and perform a syringe wash.
Final Syringe Wash	1	Washes the syringes.

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