Running the protocol

The In-Solution Digestion: Multi-Plate default protocol does the following:

•	Denaturant mixture is added to the samples.

•	Samples are denatured and reduced at elevated temperature.

•	The samples are alkylated.

•	The samples are diluted with Dilution mixture.

•	Protease is added to the samples.

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.

Stacking the wash plates and the lid for the protease plate

 

Before running the In-Solution Digestion protocol, you must stack five 96-well Greiner 650207 U-Bottom plates at AssayMAP Bravo deck location 2. The plate stacking is a requirement even if you do not run the Add Protease step or if you have fewer than four sample plates.

 

Use the Plate Stacking protocol to guide you through the plate-stacking process.

To stack the plates:

1	Label a white U-bottom plate to be used as a lid for the protease plate.

2	Label four white U-bottom plates to be used as Syringe Wash plates.

3	Fill each Syringe Wash plates with 300 µL of buffer such that the filled columns match the columns of samples in the sample plates. You can use the Single Liquid Addition v1.1 utility for this task.

4	Open the In-Solution Digestion application using one of the following methods:

•	If the Reagent Plate Setup for In-Solution Digestion: Multi-Plate page is already open, click Open In-Solution Digestion.

•	Open the App Library. (See Accessing an application.) Locate In‑Solution Digestion: Multi-Plate, and then click App.

 

The In-Solution Digestion: Multi-Plate page opens.

 

5	Click Run Plate Stacking. The Plate Stacking protocol starts.

6	Follow the instructions on the screen.

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Setting up the protocol

Before you start the protocol, make sure the appropriate selections and values are specified in the In-Solution Digestion application.

To set up the In-Solution Digestion protocol:

1	In the Application Settings area, set the following:

 

Parameter	Description
Starting Sample Volume	The volume, in microliters, of the sample in each well in the sample plate. Default: 15 Range: 0–300
Number of Sample Plates	The number of sample plates on the Bravo deck. Default: 1 Range: 1–4
Protease Storage Temperature (°C)	The temperature that should be set for the Protease plate. Default: 10 Range: 4–37

2	In the Step table:

a	Select the check box of the step that you want to perform.

b	Enter values for the selected steps:

 

Step	Description
Initial Syringe Wash	Rinses syringes with deionized water to remove potential contaminants. 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, you should specify the number of wash cycles. Increase the number of wash cycles to better clean the syringes. The step is selected by default. Wash Cycles: • Default: 3 • Range: 0–10
Add Denaturation Mixture	Adds the Denaturation Mixture to each sample plate. The syringes are washed between sample plates. Select this option to add denaturant, reductant, internal standard, or buffer to the sample plates. You may customize the components according to your experiment. The step is selected by default. Volume: • Default: 30 • Range: 1–250 Mix Cycles: • Default: 15 • Range: 0–30 Wash Cycles: • Default: 3 • Range: 0–10
Incubation (Denaturation, off deck)	Pauses the protocol run so that you can manually move the sample plates off the Bravo deck for incubation. Select this step if incubation for denaturation or reduction is required. After incubation, you must manually place the sample plates back onto the Bravo deck and resume the protocol run. The step is selected by default.
Add Alkylant	Adds the Alkylant to each sample plate. The syringes are washed between sample plates. The step is selected by default. Volume: • Default: 6 • Range: 1–250 Mix Cycles: • Default: 15 • Range: 0–30 Wash Cycles: • Default: 3 • Range: 0–10
Incubation (Alkylation, on-deck)	Incubates the lidded samples plates for the specified period (in minutes) on the Bravo deck. During incubation, alkylation occurs. The step is selected by default. Default: 45 Range: 0–180
Add Diluent Mixture	Adds the Diluent Mixture to each sample plate. The syringes are washed between sample plates. Select this step when a dilution is required to change the buffer composition of the samples or when unreacted alkylant requires quenching. The step is selected by default. Volume: • Default: 210 • Range: 1–250 Mix Cycles: • Default: 15 • Range: 0–30 Wash Cycles: • Default: 3 • Range: 0–10
Add Protease	Adds the Protease to each sample plate. The syringes are washed between sample plates. Select this step when enzymatic digestion is required. The step is selected by default. Volume: • Default: 9 • Range: 1–250 Mix Cycles: • Default: 15 • Range: 0–30 Wash Cycles: • Default: 3 • Range: 0–10
Final Syringe Wash	Rinses the syringes with deionized water the specified number of cycles. Select this step to clean the syringes thoroughly before running a different assay. Increase the number of wash cycles to better clean the syringes. The step is selected by default. Wash Cycles: • Default: 3 • Range: 0–10

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

 

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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 decrease the volumes.

 

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Starting the protocol run

To start the protocol run:

1	Ensure that the physical layout on the AssayMAP Bravo deck matches the Deck Layout and the Labware Table in the In-Solution Digestion application.

At deck location 7, ensure the Denaturation Mixture plate is stacked atop the Alkylation plate.

Note: The Reagent Plate Setup protocol stacks the labware at deck location 7 automatically. If you prepared the reagent plates manually, you must stack the plates manually at deck location 7.

 

The deck layout for the In-Solution Digestion protocol must always be set up exactly as shown in the Deck Layout and Labware Table. Do not remove a reagent plate from the deck even if the reagent is not being used. The protocol requires these labware to be in their defined positions to run properly. Instead, use an empty microplate as a place holder. The only optional labware in the In-Solution Digestion protocol are the sample plates at deck locations 6, 8, and 9. The number of sample plates can vary according to the Number of Sample Plates parameter in the protocol.

 

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

 

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 In-Solution Digestion. The protocol run starts.

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

 

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.

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 AssayMAP Bravo deck.

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

•	Run the System Startup/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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