Running the protocol

The Fractionation protocol:

•	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 peptides 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 seating station.

Before you start

Ensure that you:

•	Prepare the reagents. See Preparing the reagents.

•	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.

 

Stacking Collection plates and Elution Buffer plates

You must prepare stacks of pre-filled Elution Buffer plates and empty Fraction Collection plates on the Bravo deck before running the Fractionation protocol. Use the Plate Stacking protocol to stack the plates.

 

You should always use the Plate Stacking protocol to stack the plates. Stacking the labware manually can produce misaligned stacks, resulting in crashes during the protocol run.

 

 

Do not use labware that have been repeatedly heat sealed or subjected to extreme heat or cold. Warped labware can cause imprecise stacking, potentially causing head crashes during the protocol run.

 

To run the Stack Plates protocol:

1	Label the empty Fraction Collection plates.

2	Label the empty Elution Buffer plates, and then fill them with the volume of Elution Buffer as specified in the Fractionation Calculator.

3	Open the App Library. See Accessing an application.

4	Locate Fractionation, and then click App.

 

The Fractionation application opens.

 

5	In the A. Run Plate Stacking Utility area, specify the Number of Fractions that will be collected during the Fractionation protocol run. The value you specify must match the number Elution Buffer plates you are using.

Range: 0–6

Default: 0

6	Click Run Plate Stacking. The Stack Plates protocol starts.

7	Follow the instructions on the screen.

 

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Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Fractionation application.

To set up the Fractionation protocol:

1	Optional. In the Fractionation application, click Restore Defaults to set all Application Settings to their defaults.

2	In the B. Application Settings area, specify the Number of Full Columns 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: 1

 

If the column 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 software specifies 10 columns, but only 1 column of cartridges are in the seating station, the head will apply 10 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

 

 

If the column selection in the software 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 software specifies 1 column, but 10 columns of cartridges are in the seating station, the head will apply 1/10th 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.

 

3	In the Application Settings table, select the check boxes of the steps that you want to perform and enter the values for the selected steps. For details, see Application Settings table.

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

 

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

 

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

 

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Application Settings table

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

 

Step	Description
Initial Syringe Wash	Rinses syringes with deionized water in the specified number of cycles. Select the Initial Syringe Wash step to clean the syringes. Clear the check box if you are certain that the syringes are clean. If you select this step, specify the number of wash cycles. Increase the number of wash cycles to better clean the syringes. However, be aware that increasing the number of cycles increases the time of the assay and consumption of reagents, and affects wear on the syringes. This step is selected by default. Wash Cycles: Default: 3
Prime	Primes the cartridges with Priming Buffer. IMPORTANT Cartridges ship dry and therefore contain air entrained in the cartridge bed. Failure to prime cartridges can prevent access of the sample and buffers to parts of the bed, resulting in reduced capacity and poor reproducibility. Always perform the Prime step at the beginning of a run. This step is selected by default. Volume: Default: 100 Flow Rate: Default: 300 Wash Cycles: Default: 1
Equilibrate	Exchanges the Priming Buffer with the Equilibration Buffer within the resin bed. You should always equilibrate the cartridge bed with a buffer that establishes optimal chemical conditions for sample loading. This step is selected by default. Volume: Default: 50 Flow Rate: Default: 10 Wash Cycles: Default: 1
Load Sample	Loads the samples on the cartridges, and then performs the syringe wash cycles at the wash station. Specify the sample volume and flow rate, and the number of syringe wash cycles to be performed following sample loading. Ensure that each sample well has the recommended overage (labware dependent) to prevent air from getting introduced into the cartridge. This step is selected by default. Volume: Default: 100 Flow Rate: Default: 5 Wash Cycles: Default: 3
Collect Flow Through	Dispenses the sample flow-through into Flow Through Collection. The option is only applicable if you select the Load Samples step. Select the option if you want to collect the sample flow-through for downstream processing or analysis. Clear the check box if you want dispense the sample flow-through into the Organic Waste plate. This step is selected by default.
Cup Wash	Washes the cartridge cup using the Equilibration Buffer. Select this step to remove the small amount of sample that remains in the cup to improve the efficiency of the Internal Cartridge Wash. This step is selected by default. Volume: Default: 50 Wash Cycles: Default: 1
Internal Cartridge Wash	Removes non-specifically bound molecules from the cartridge resin using the Equilibration Buffer. Select this step to minimize binding of weakly bound and non-specifically bound species from the resin in the cartridge and to remove non-target elements, which could possibly contaminate the elution. This step is selected by default. Volume: Default: 25 Flow Rate: Default: 5 Wash Cycles: Default: 3
Collect Flow Through	Dispenses the buffer from the Internal Cartridge Wash step into Flow Through Collection (deck location 8). The option is only applicable if you select the Internal Cartridge Wash step. Select the option if you want to collect the flow-through and combine it with the sample flow-through from the Load Samples step for downstream processing or analysis. Clear the check box if you want to dispense the buffer into the Organic Waste plate. This step is selected by default.
Predispense Elution Buffer	Adds the Elution Buffer to each fraction plate before collecting the fraction. This step is helpful in collecting small fraction volumes. This step is not selected by default. Volume: Default: 15
Elute Fraction 1-6	Allows the independent selection of elution volumes and flow rates for each fraction to be collected. The number of wash cycles between each fraction can also be specified. The number of fractions to be eluted from the cartridges and collected in Fraction Collection plates is determined by the value selected in the Number of Fractions list. Volume: Default: 25 Flow Rate: Default: 5 Wash Cycles: Default: 1
Final Syringe Wash	The option to rinse syringes with deionized water the specified number of cycles at the wash station. Select this step to clean the syringes thoroughly before running a different assay. The step is selected by default. This step is selected by default. Wash Cycles: Default: 3

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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.

 

 

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Starting the protocol run

To start the protocol run:

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

•	The physical layout on the Bravo deck matches the display 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. The default protocol with 100-µL samples and 5-µL/min flow rate should take approximately 55 minutes to complete.

 

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, 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 Fractionation 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.

 

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Cleaning up

When the protocol run is finished, make sure you:

•	Remove all labware from the Bravo deck.

•	Discard solutions, organic waste, and used labware following appropriate waste disposal procedures.

•	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.

 

 

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