Running the protocol

The Protein Cleanup v2.0 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 protein 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 buffers. See Preparing the solutions.

•	Prepare the samples. See Preparing the samples.

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

•	Transfer the cartridges to the 96AM 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 Protein Cleanup application.

To set up the protocol:

1	Open the App Library. See Accessing an application.

2	Locate Protein Cleanup v2.0, and then click App.

 

The Protein Cleanup v2.0 application opens.

 

3	Under Select Method, click , locate and select the method file, and then click Load to display all the settings associated with the selected method.

The Status area displays the loading status.

 

Note: Protein Cleanup v2.0 methods are saved in the C:\VWorks Workspace\Methods\Protein Cleanup v2.0 folder. Agilent provides a method with default settings for each cartridge size.

 

Ensure that the method is for the AssayMAP cartridge size you are using: 5 µL or 25 µL. The names of the methods that Agilent provides have the cartridge size as a prefix.

 

To modify the method, proceed to step 4. Otherwise, go to Starting the protocol run.

 

 

 

4	In the 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 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 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. 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 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. To obtain expected instrument performance, 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.

 

5	In the Application Settings table:

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

b	Enter values for the selected steps.

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

7	To save the method, specify the method Name in the Save Method area.

Agilent recommends that you use the cartridge size (5 µL or 25 µL) as a prefix to the name.

If applicable, select Overwrite if Name Exists, and then click Save to save the settings in a method file.

 

 

 

 

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Application Settings table

The following figure shows the default settings for each cartridge size, and the 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.

Figure. Default settings for each cartridge size

 

5 µL cartridge	25 µL cartridge

Table Descriptions of each step

 

Steps	Description
Initial Syringe Wash	Rinses the syringes 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: 3
Prime	Primes the cartridges with the Priming & Syringe Wash Buffer. 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. Always perform the Prime step at the beginning of a run. Specify the volume of buffer, the flow rate, and the number of syringe wash cycles to be performed following priming. This step is selected by default. Volume (µL): • Default for 5 µL cartridge: 100 • Default for 25 µL cartridge: 250 Flow Rate (µL/min): Default: 300 Wash Cycles: Default: 1
Equilibrate	Ensures that the resin bed has been fully equilibrated in preparation for sample loading by exchanging the Priming Buffer with the Equilibration Buffer within the resin bed. Always perform the Equilibrate step after cartridge priming to establish optimum sample loading conditions. Specify the volume of Equilibration & Cartridge Wash Buffer to use, the flow rate, and the number of syringe wash cycles to be performed following equilibration. This step is selected by default. Volume (µL): • Default for 5 µL cartridge: 50 • Default for 25 µL cartridge: 250 Note: Setting Volume at 0 skips all Equilibrate tasks except for syringe washing. Flow Rate (µL/min): Default: 10 Wash Cycles: Default: 1
Load Samples	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. This step is selected by default. Volume (µL): Default: 100 Note: Setting Volume at 0 skips all Load Samples tasks except for syringe washing. 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 in the Organic Waste plate. This step is not selected by default.
Cup Wash	Washes the cups of the parked cartridges using Equilibration & Cartridge Wash Buffer, 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. Specify the buffer volume for the cup wash and the number of wash cycles. This step is selected by default. Volume (µL): Default: 25 Wash Cycles: Default: 3 Note: Each wash cycle consists of a cup wash followed by syringe wash.
Internal Cartridge Wash	Removes non-specifically bound molecules from the cartridge resin using the Equilibration & Cartridge Wash Buffer. A 25-µL sample chase with Equilibration & Cartridge Wash Buffer occurs at the Load Samples flow rate before the Internal Cartridge Wash step. Select this step to minimize non-target elements from the sample matrix in the cartridge which could possibly contaminate the eluate. This step is selected by default. Volume (µL): • Default for 5 µL cartridge: 50 • Default for 25 µL cartridge: 250 Note: Setting Volume at 0 skips all Internal Cartridge Wash tasks except for syringe washing. Flow Rate (µL/min): Default: 10 Wash Cycles: Default: 3
Collect Flow Through	Select this option if you want to collect the flow-through from Internal Cartridge Wash in the Flow Through Collection plate. Clear the check box if you want dispense the flow-through in the Organic Waste plate. This step is not selected by default.
Stringent Syringe Wash	Washes the syringes with the Priming & 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 Note: Setting Volume at 0 skips the Stringent Syringe Wash step. Wash Cycles: Default: 2
Elute	Elutes bound proteins from the cartridges. Select this step to remove the bound protein from cartridges. This step is selected by default. Volume (µL): • Default for 5 µL cartridge: 25 • Default for 25 µL cartridge: 125 Note: Setting Volume at 0 skips all Elute tasks except for syringe washing. Flow Rate (µL/min): Default: 5 Wash Cycles: Default: 1
Eluate Discard	Discards the specified volume from the beginning of the elution step. The volume can equal, but not exceed the Elute volume. Select this option if you want to minimize the volume of eluate. This step is not selected by default. Volume (µL): Default: 0 Note: The protocol uses the flow rate value that you specified for the Elute step.
Add to Flow Through	Adds the volume specified in the Eluate Discard to the Flow Through Collection plate. If you do not select this step, the Eluate Discard volume is dispensed in the Organic Waste plate. This step is not selected by default.
Existing Collection Volume	Defines the volume of buffer in each well of the Eluate Collection plate before the start of the protocol. This value is used to calculate the sample mixing volume and to adjust the well-bottom offset for elution. Volume (µL): Default: 0
Final Syringe Wash	Washes the syringes 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: 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 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 Protein Cleanup 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.

•	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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