Running the protocol

The Affinity Purification: Aspiration Mode protocol does the following:

•	Washes the syringes.

•	Primes and equilibrates the cartridges to prepare for sample loading.

•	Loads the samples onto the cartridges.

•	Removes non-specific binding molecules from the cartridges.

•	Elutes the analyte from the cartridges.

For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the Cartridge & Tip Seating Station.

Before you start

Ensure that you:

•	Prepare the reagents. See Preparing the solutions.

•	Prepare the samples. See Preparing the samples.

•	Run the Startup utility to prepare the AssayMAP Bravo Platform for the run. See System Startup/Shutdown v2.2.

•	Transfer the cartridges to the Cartridge & Tip Seating Station. See Cartridge Transfer v1.1.

 

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 bed, resulting in reduced capacity and poor reproducibility.

 

 

Do not allow wetted cartridges to dry out. Agilent Technologies does not guarantee performance of stored cartridges following equilibration. See Cartridge use and storage guidelines.

 

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Affinity Purification: Aspiration Mode application.

To set up the protocol:

1	Open the App Library. See Accessing an application.

2	Locate Affinity Purification: Aspiration Mode v2.0, and then click App.

 

The Affinity Purification: Aspiration Mode application opens.

 

3	Optional. Click Restore Defaults to set all Application Settings to their defaults.

4	In the Labware Table area, select the labware you are using for the protocol run.

5	In the Application Settings area, specify the Number of Cartridges to use during the run. The position of the columns of cartridge in the tip seating station must match the positions of the samples and solutions in the plates on the deck.

Range: 1–12

Default: 8

 

Ensure that you specify the number of cartridges for the Affinity Purification: Aspiration Mode app instead of the number of columns, which is the setting used in the other AssayMAP apps. Also, make sure to design experiments in a column format to avoid droplets falling on the deck as described in Planning the microplate setup.

 

 

If the cartridge selection is greater than the actual number of cartridges 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 form specifies 96 cartridges, but only 8 cartridges are in the seating station, the head will apply 12 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

 

 

If the cartridge selection is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the form specifies 8 cartridges, but 96 cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. To prevent potential equipment damage, ensure that the column selection is correct.

 

 

Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

 

6	In the Application Settings table, do the following. For details, see Application Settings table:

a	Select the check boxes of the steps that you want to perform.

b	Enter values for the selected steps.

7	Optional. When you are finished, click Save Settings.

 

Return to top

Application Settings table

The following table gives a brief description of each setting. For details, including the full and practical ranges of values for a given setting, see the Assay development guidelines and protocol notes.

 

 

Step	Description
Initial Syringe Wash	Rinses the syringes at the wash station with deionized water to remove potential contaminants. Specify the number of wash cycles. Clear the check box if you are certain that the syringes are clean. This step is selected by default. Wash Cycles: Default: 5
Prime	Uses the Priming & Equilibration Buffer to ensure entrapped air is removed from the cartridges. IMPORTANT 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 bed, resulting in reduced capacity and poor reproducibility. This step fills the syringes with sufficient Priming & Equilibration Buffer to wet the inlet seal (10 μL) and perform the Prime (100 μL) and Equilibration steps (50 μL default). Before mounting the cartridges on the syringe probes, the Bravo 96AM head dispenses 10 μL into each cartridge cup to prewet the inlet seal to ensure no air bubbles remain trapped in this small area. After mounting the cartridges on the probes, the head moves by a fixed offset between the chimneys in the wash station to dispense the priming buffer through the cartridges to waste. Always perform the Prime step at the beginning of a run. The volume, flow rate, and wash cycle setting for this step are selected by default: • Volume (µL): 100 • Flow Rate (µL/min): 300 • Wash Cycles: 1
Equilibrate	Ensures that the resin bed has been fully equilibrated in preparation for sample loading. Always perform the Equilibrate step after cartridge priming to establish optimum sample loading conditions. Specify the volume of Priming & Equilibration Buffer to use and the flow rate. This step is selected by default. Volume (µL): If you select Prime and Equilibrate, the system calculates the volume required to perform both steps and aspirates it during the Prime step. Default: 50 Flow Rate (µL/min): Default: 25
Load Samples	Loads the samples on the cartridges. This step aspirates samples through the mounted cartridges into the syringes, performs an external cartridge tip wash at the wash station, and then aspirates a 5-µL chase of Priming & Equilibration Buffer through the mounted cartridges. After parking the cartridges, the flow-through is dispensed into the wash station (default) or into Flow Through Collection. Specify the sample volume and flow rate, and the number of syringe wash cycles to be performed following sample loading. This step is selected by default. Volume (µL): Default: 50 Flow Rate (µL/min): Default: 5 Wash Cycles: Default: 3
Collect Flow Through	Select this option if you want to collect the sample flow-through in the Flow Through Collection plate. Clear the check box if you want to dispense the sample flow-through into the wash station. This step is not selected by default.
Cup Wash 1	Washes the cups of the parked cartridges using Cartridge Wash Buffer 1, and then performs a syringe wash at the wash station. Select this step to remove the small amount of sample that remains in the cup to improve the efficiency of the Internal Cartridge Wash 1. Specify the buffer volume for the cup wash and the number of wash cycles. This step is selected by default. Volume (µL): Default: 50 Wash Cycles: Default: 2 Note: Each wash cycle consists of a cup wash followed by syringe wash.
Internal Cartridge Wash 1	Uses Cartridge Wash Buffer 1 to wash non-specifically bound molecules from the cartridges. This step aspirates Cartridge Wash Buffer 1 through the mounted cartridges into the syringes, performs an external cartridge tip wash at the wash station. After parking the cartridges, the flow-through is dispensed into the wash station. This step is selected by default. Volume (µL): Default: 50 Flow Rate (µL/min): Default: 25 Wash Cycles: Default: 5
Cup Wash 2	Washes the cups of the parked cartridges using Cartridge Wash Buffer 2, and then performs a syringe wash at the wash station. Select this step to remove the small amount of Cartridge Wash Buffer 1 that remains in the cup to improve the efficiency of Internal Cartridge Wash 2. This step is not selected by default. Volume (µL): Default: 50 Wash Cycles: Default: 1 Note: Each wash cycle consists of a cup wash followed by syringe wash.
Internal Cartridge Wash 2	Uses Cartridge Wash Buffer 2 to wash non-target elements from the cartridge resin. This step aspirates Cartridge Wash Buffer 2 through the mounted cartridges into the syringes, and then performs an external cartridge tip wash at the wash station. After parking the cartridges, the flow-through is dispensed into the wash station. Select this step to minimize non-target elements from the sample matrix in the cartridge which could possibly contaminate the eluate. This step is not selected by default. Volume (µL): Default: 50 Flow Rate (µL/min): Default: 25 Wash Cycles: Default: 5
Stringent Syringe Wash	Washes the syringes with the Elution & Syringe Wash Buffer followed by a syringe wash at the wash station. Select this step to rigorously clean the syringes before the elution step. This step is selected by default. Volume (µL): Default: 50 Wash Cycles: Default: 5
Elute	Elutes bound analytes from the cartridges using the Elution & Syringe Wash Buffer, and then performs syringe washes at the wash station. Select this step to remove the bound analyte from the cartridges. This step is selected by default. Volume (µL): Default: 50 Flow Rate (µL/min): Default: 10
Re-Equilibrate	Uses the Priming & Equilibration Buffer to return the cartridge to a neutral pH solution. This step aspirates buffer through the mounted cartridges into the syringes, removes the cartridges, and then dispenses the liquid into the wash station. This step is selected by default. Volume (µL): Default: 50 Flow Rate (µL/min): Default: 25
Final Syringe Wash	Washes the syringes with deionized water the specified number of cycles at the wash station. Select this step to clean the syringes. This step is selected by default. Wash Cycles: Default: 5

 

Return to top

About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles.

You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 µL/min and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

 

The protocol will not run if cartridges are missing. You can use resin-free cartridges instead of packed cartridges for mock runs.

 

 

Return to top

Starting the protocol run

To start the protocol run:

1	Review the selections and value specifications in the protocol to confirm they are correct. Make sure:

•	The physical layout on the Bravo deck matches the graphic shown in the Deck Layout area.

•	In the Labware Table area, the selections reflect the labware you are using for the protocol run.

2	Make sure the labware are properly seated on the Bravo deck.

 

Use only the labware specified, and place them at the AssayMAP Bravo deck locations specified in the form. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

 

 

Improperly seated labware can cause a hardware collision, resulting in equipment damage. Ensure that all labware are properly seated within the alignment features of their respective platepads.

 

3	Click Run Protocol.

An error message appears if the software detects an error in the form settings, for example, if an entered value is outside the limits set for a field. If this happens, follow the directions in the message to select Pause and Diagnose, and then select Abort process. After setting the appropriate values, click Run Protocol to restart the run.

Note: For the range of acceptable values for a given setting, see the Assay development guidelines and protocol notes.

To monitor the progress of the run, check the Status box.

 

After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol.

 

To stop a run in an emergency, use the hardware Emergency Stop button.

 

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses.

 

Protocols should not be paused to change the settings. However, if you need to change a setting in a running protocol, click Pause during the last task of a step, and then click Abort process in the dialog box that appears. Change the setting, and then clear the check boxes of the steps in the Affinity Purification: Aspiration Mode application that have been completed successfully. Restart the protocol to complete the remaining steps.

 

To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

 

Return to top

Cleaning up

When the protocol run is finished, make sure you:

•	Remove all labware from the Bravo deck.

•	Run the System Shutdown utility. See System Startup/Shutdown v2.2.

 

Make sure you discard the chemical waste and used labware according to your lab’s waste disposal procedures and in compliance with all local, state, and federal safety regulations.

 

 

Return to top