Assay development guidelines and protocol notes

This topic explains each step of the protocol so that you can optimize the 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 reagents and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

 

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 first stage in the protocol is to set up the system and wash the syringes in the Bravo 96AM Head to ensure no carryover from previous experiments remains. The Initial Syringe Wash step removes any potential contaminants from the syringes. Each cycle of the Initial Syringe Wash step 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. In the default protocol, this step is selected.

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 cartridges, the Priming Buffer requires that the solution contain at least 50% organic solvent, typically, acetonitrile. Higher concentrations of organic solvent are also acceptable.

The Prime step aspirates the specified volume of Priming Buffer (deck location 4) 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 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.

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 slower 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 does not permit the Priming Buffer to fully wet the resin.
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.

Strip Metal step

The Strip Metal step removes the metal coordinated to the Fe(III)-NTA resin by using a strong chelator, such as EDTA. Complete removal of the bound metal from the resin bed is critical so that a different metal can be bound at the maximum metal-binding capacity of the cartridge.

The Strip Metal step aspirates the specified volume of Metal Stripping Buffer (deck location 5) into the syringes, and then mounts the cartridges at the seating station (deck location 2). The buffer is dispensed through cartridges at the specified flow rate into the Waste plate (deck location 3), or into the Flow Through Collection plate (deck location 7) if the Collect Flow Through step is selected. During an external cartridge tip wash, the cartridge tips dip into the wash station chimneys (deck location 1) to remove any residual liquid 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.

Always perform the Strip Metal step after cartridge priming to remove bound metal from the Fe(III)-NTA resin. In the default protocol, this step is selected.

Table Setting the Strip Metal step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 100 Practical: 50–100 Range: 0–250	The default volume, 100 µL, is equal to 20-column volumes, which should be sufficient for complete removal of bound metal using a sufficiently concentrated chelator in the Metal Stripping Buffer. Note: Setting the Volume at 0 skips all Strip Metal step tasks, except syringe washing.
Flow Rate (µL/min)	Default: 5 Practical: 2–15 Range: 0.5–500	A flow rate slower than < 2 µL/min will likely have no benefit, but will increase the total assay time. A flow rate > 15 µL/min using the default volume might not permit full penetration into the pores of the resin across the full length of the cartridge bed leaving residual metal on the cartridge.
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 (Strip Metal step)

Note: The Collect Flow Through step is an option only if the Strip Metal step is conducted.

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

If the Strip Metal step is conducted, but the Collect Flow Through step is not selected, the flow-through is dispensed directly into the 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 flow-through generated during the Strip Metal step for downstream processing or for analysis.

Cup Wash 1 step

The Cup Wash 1 step removes the small volume of residual liquid that might remain above the upper cartridge membrane after the Strip Metal step. The Cup Wash step effectively rinses the cup portion of AssayMAP cartridges with buffer. The Cartridge Wash Buffer 1 (deck location 6) is aspirated into the syringes, and then dispensed into the cartridge cups (deck location 2). This liquid, plus any residual liquid, 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 Waste plate (deck location 3), and then washing the syringes at the wash station (deck location 1).

Select the Cup Wash 1 step if residual liquid remains in the cartridge cups from the Strip Metal step. In the default protocol, this step is selected.

Table Setting the Cup Wash 1 step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 25 Practical: 25–50 Range: 0–100	This parameter specifies the volume of Cartridge Wash Buffer 1 to dispense into the cartridge cup. A volume < 10 µL might be insufficient for cup washing. A volume > 50 µL might offer little benefit. Note: Setting the Volume at 0 skips all tasks associated with the Cup Wash 1 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 1 step

The Internal Cartridge Wash 1 step aspirates Cartridge Wash Buffer 1 (deck location 6) 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 Waste plate (deck location 3), or into the Flow Through Collection plate (deck location 7) if the Collect Flow Through step is selected. 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 Cartridge Wash Buffer 1 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.

Select the Internal Cartridge Wash 1 step to flush away any remaining Metal Stripping Buffer and prepare the cartridge with a buffer compatible with the Metal Reagent in the Load Metal step. In the default protocol, this step is selected.

Table Setting the Internal Cartridge Wash 1step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 50 Practical: 50–100 Range: 0–250	This parameter specifies the volume of Cartridge Wash Buffer 1 to dispense through cartridges to wash the resin bed. Note: Setting the Volume at 0 skips all tasks associated with the Internal Cartridge Wash 1 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 penetrate through the pores in the resin across the full length of the cartridge bed, which can lead to 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 (Internal Cartridge Wash 1 step)

Note: The Collect Flow Through step is an option only if the Internal Cartridge Wash 1 step is conducted.

The Collect Flow Through step elutes the wash flow-through from the Internal Cartridge Wash 1 step directly into the Flow Through Collection plate (deck location 7). 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 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 1 step. In the default protocol, this step is not selected.

Load Metal Reagent step

The Load Metal Reagent step aspirates the specified volume of Metal Reagent (deck location 9) into the syringes, performs an external syringe wash at the wash station (deck location 1) to remove any Metal Reagent remaining on the outside of the probes, and then mounts the cartridges at the seating station (deck location 2). The Metal Reagent is dispensed through the cartridges at the specified flow rate into the Waste plate (deck location 3), or into the Flow Through Collection plate (deck location 7) if the Collect Flow Through step is selected.

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 Metal Reagent 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.

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

Table Setting the Load Metal Reagent step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 100 Practical: 50–150 Range: 0–250	The volume of the Metal Reagent should be balanced with the concentration of the Metal Reagent and the binding capacity of the cartridge. Note: Setting the Volume at 0 skips all Load Metal Reagent step tasks, except syringe washing.
Flow Rate (µL/min)	Default: 5 Practical: 2–15 Range: 0.5–500	A flow rate < 2 µL/min will likely have no benefit, but will increase the total assay time. A flow rate > 15 µL/min using the default volume might not permit full penetration into the pores of the resin across the full length of the cartridge bed, leaving portions of uncharged NTA in the resin bed, which would reduce binding capacity.
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 (Load Metal Reagent step)

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

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

If the Load Metal Reagent step is conducted, but the Collect Flow Through step is not selected, the flow-through is dispensed directly into the 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 flow-through generated during the Load Metal Reagent step for downstream processing or for analysis. In the default protocol, this step is not selected.

Cup Wash 2 step

The Cup Wash 2 step removes the small volume of residual liquid that might remain above the upper cartridge membrane after the Load Metal Reagent step. The Cup Wash 2 step effectively rinses the cup portion of AssayMAP cartridges with buffer. The Cartridge Wash Buffer 2 (deck location 8) is aspirated into the syringes, and then dispensed into the cartridge cups (deck location 2). This liquid, plus any residual liquid, 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 Waste plate (deck location 3), and then washing the syringes at the wash station (deck location 1).

Select the Cup Wash 2 step if residual liquid remains in the cartridge cups from the Load Metal Reagent step. In the default protocol, this step is selected.

Table Setting the Cup Wash 2 step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 25 Practical: 25–50 Range: 0–100	This parameter specifies the volume of Cartridge Wash Buffer 2 to dispense into the cartridge cup. A volume < 10 µL might be insufficient for cup washing. A volume > 50 µL might offer little benefit. Note: Setting the Volume at 0 skips all tasks associated with the Cup Wash 2 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 2 step

The Internal Cartridge Wash 2 step aspirates Cartridge Wash Buffer 2 (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 Waste plate (deck location 3), or into the Flow Through Collection plate (deck location 7) if the Collect Flow Through step is selected. 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 Cartridge Wash Buffer 2 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.

Select the Internal Cartridge Wash 2 step to flush away any remaining Metal Reagent and prepare the cartridge with a buffer compatible with immediate use or short-term storage. In the default protocol, this step is selected.

Table Setting the Internal Cartridge Wash 2 step parameters

 

Parameter	Value	Notes
Volume (µL)	Default: 50 Practical: 50–100 Range: 0–250	This parameter specifies the volume of Cartridge Wash Buffer 2 to dispense through cartridges to wash the resin bed. Note: Setting the Volume at 0 skips all tasks associated with the Internal Cartridge Wash 2 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 penetrate through the pores in the resin across the full length of the cartridge bed, which can lead to 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 2 step)

Note: The Collect Flow Through step is an option only if the Internal Cartridge Wash 2 step is conducted.

The Collect Flow Through step dispenses the wash flow-through from the Internal Cartridge Wash 2 step directly into the Flow Through Collection plate (deck location 7). If the Internal Cartridge Wash 2 step is conducted, but the Collect Flow Through step is not selected, the wash flow-through is dispensed directly into the 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 2 step. In the default protocol, this step is not selected.

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 protocol when the protocol defaults are used. Changing the selections or deselecting options will alter the movements. The default protocol takes approximately 60 minutes to complete.

 

Protocol step	Head moves to deck location...	Action
Run Cartridge Transfer utility	6	Mounts one column of Fe(III)-NTA 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 IMAC Cartridge Customization 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	4	Aspirates the Priming Buffer into the syringes.
	2	Mounts the cartridges onto the head.
	3	Dispenses the Priming Buffer through the cartridges into the Waste plate to prime the cartridges.
	2	Parks the cartridges.
	1	Washes the syringes for 1 cycle.
Strip Metal	5	Aspirates the Metal Stripping Buffer into the syringes.
	2	Mounts the cartridges on the head.
	3	Dispenses the Metal Stripping Buffer through the cartridges into the Waste 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 1	6	Aspirates the Cartridge Wash Buffer 1 into the syringes.
	2	Washes the cartridge cups.
	3	Dispenses the Cartridge Wash buffer 1 into the Waste plate.
	1	Washes the syringes for 1 cycle.
Internal Cartridge Wash 1	6	Aspirates the Cartridge Wash Buffer 1 into the syringes.
	2	Mounts the cartridges.
	3	Dispenses the Cartridge Wash Buffer 1 through the cartridges into the 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.
Load Metal Reagent	9	Aspirates the Metal Reagent into the syringes.
	1	Washes the exterior of the syringe probes at the wash station.
	2	Mounts the cartridges on the head.
	3	Dispenses the Metal Reagent through the cartridges into the Waste 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 2	8	Aspirates the Cartridge Wash Buffer 2 into the syringes.
	2	Washes the cartridge cups.
	3	Dispenses the Cartridge Wash buffer 2 into the Waste plate.
	1	Washes the syringes for 1 cycle.
Internal Cartridge Wash 2	8	Aspirates the Cartridge Wash Buffer 2 into the syringes.
	2	Mounts the cartridges.
	3	Dispenses the Cartridge Wash Buffer 2 through the cartridges into the 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.
Final Syringe Wash	1	Washes the syringes for 3 cycles.

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