Preparing the reagents

The In-Solution Digestion protocol accommodates a wide range of digestion chemistries so that previously optimized conditions can be easily transferred to this automated platform. Therefore the reagents described in this topic are categories of reagents used in the assay rather than specific recommendations.

The following reagents are required for the In-Solution Digestion protocol:

 

Reagent	# of plates required	Description
Syringe Wash Buffer	4	This is an optional solution used to solubilize and wash away protein that may have adsorbed onto the probes and syringes. The use of 50 mM NaOH has been found to be a suitable stringent syringe wash solution for several proteins. However, other protein-solubilizing solutions may be used depending on the specific protein sample inputs. Use the Single Liquid Addition v1.1 utility to transfer 300 µL per well of the wash solution to the four Greiner 96-Well Round Bottom, White Plates that will be stacked on deck location 2 prior to running the In-Solution Digestion protocol. During the protocol run, these plates serve two purposes. The plates are moved on top of the sample plates and serve as lids to protect the samples from light during the alkylation step of the protocol. The plates are also used as reservoirs for the syringe wash solution which is used to wash the syringes to prevent carry-over between sample loading steps.
Master Reagents	1	There are three master mix reagents: Denaturation mixture, alkylant, and protease. The three reagents are placed in the reagent set up plate which serves as the source plate for distributing these reagents into the plates used during the In-Solution Digestion protocol. Use the Reagent Plate Setup protocol to dispense these three reagents to dedicated reagent plates. Fill an ABgene 1.2 mL Deep-Well Plate with the Master Reagent. See Running the Reagent Plate Setup protocol.
Diluent Mixture	1	This reagent is placed in an ABgene 1.2-ml deep-well plate which is placed at deck location 3 during the In-Solution Digestion protocol run. This solution is used to dilute the sample following denaturation, reduction, alkylation and before enzyme addition. The solution typically contains deionized water, 1.0 M Tris at pH 8.1, and 0.5 M TCEP. The volume per well required for the Diluent Mixture and the composition of the solution is determined using the Reagent Volume Calculator.

Use the supplied Reagent Volume Calculator to determine the optimal reagent formulations. Run the Reagent Plate Setup protocol using the calculated values to dispense the reagents into the appropriate labware.

 

A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

 

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

About the Reagent Volume Calculator for In-Solution Digestion

The Reagent Volume Calculator is a Microsoft Excel worksheet that:

•	Allows you to enter values such as the number of samples to process.

•	Calculates reagent needs and recipe volumes based on your input.

•	Takes into consideration overages.

The calculator consists of the following worksheets:

 

Worksheet	Description
Reaction Setup	Allows you to enter information about the samples, denaturant and reductant, alkylant, dilution, and digestion. Based on your input, the calculator determines the reagent concentrations and volumes. You use the calculated volumes when running the Reagent Plate Setup protocol. See Dispensing the reagents.
Reagent Prep	Displays the volumes and composition of the reagents required for the in-solution assay based on the input provided in the Reaction Setup section of the calculator. The regent volumes are automatically adjusted for required overages.
Automated Plate Setup	Displays the layout of the Master Reagent plate and the Diluent Mixture plate, including the volumes of the reagents that must be transferred into the designated wells when running the Reagent Plate Setup protocol. See Dispensing the reagents.
Manual Plate Setup	Displays the layout of the Denaturation Mixture plate, Alkylant plate, Protease plate, and Diluent Mixture plate, including the volumes of the reagents required for the plates used in the In-Solution Digestion protocol in case you prefer to manually pipette these reagents into the plates rather than use the Reagent Plate Setup protocol.

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Opening the Reagent Volume Calculator for In-Solution Digestion: Multi-Plate

To open the Reagent Volume Calculator:

1	Open the App Library. See Accessing an application.

2	Locate In-Solution Digestion: Multi-Plate, and then click Calculator. Microsoft Excel starts and displays the calculator.

3	Notice that you can access the four worksheets using the tabs at the bottom of the screen:

•	Reaction Setup

•	Reagent Prep

•	Automated Plate Setup

•	Manual Plate Setup

 

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Entering values in the Reaction Setup worksheet

The Reaction Setup worksheet consists of three tables:

•	Reaction Setup

•	Reactant Concentration Summary

•	In-Solution Digest form inputs

Based on the values you supply in the green boxes, the software will calculate and update the read-only values (gray boxes) in the rest of the worksheet.

The boxes for which you are allowed to enter values should remain green after you enter a value. If you enter a value that meets only some of the requirements, the box becomes yellow. If you enter a value that does not meet any requirement, the box becomes red.

To enter values in the Reaction Setup worksheet:

1	In the Reaction Setup table, type the values in the green boxes in the 1. Sample Input area:

 

 

Field	Description
Number of Samples	The number of samples you want to process. The number of samples should be a multiple of 8. Note: If the number of samples is not a multiple of 8, excess reagent consumption will occur. See Total Number of Sample Columns. Range: 8–384 Default: 96
Total Number of Sample Columns	Read only. The calculator converts the Number of Samples to the Total Number of Sample Columns by rounding up to the nearest multiple of 8. Range: 1–48 Default: 12
Number of Sample Plates	The number of labware containing the samples you want to process. To minimize the volume of reagent consumed, specify a greater number of sample plates where fewer wells are used in each plate. This requires fewer syringes during the liquid transfer, which minimizes reagent consumption. To minimize the number of labware used, specify fewer number of sample plates. For example, place eight columns of samples on one plate. Because more syringes are used during the liquid transfer, more reagents are consumed. To assess which scenario works best, use the Reagent Prep worksheet. Range: 1–4 Default: 2
Number of Columns per Sample Plate	Read only. The number of columns that occupy each sample plate. The value is calculated using the Total Number of Sample Columns and the Number of Sample Plates. Range: 1–12 Default: 6
Starting Column Number on Sample Plate	The column at which the samples begin in the sample plate. Note: The samples can begin in any column in the sample plate. Make sure the columns in the plates are contiguous. You cannot skip a column. If you have multiple sample plates, make sure the samples begins at the same column across all the plates. Range: 1–12 Default: 1
Sample Volume, µL	The volume of the samples in each well. IMPORTANT Make sure the labware you are using can accommodate the volume you specify. You can start with the default value, 15 µL, because it satisfies a typical set of reaction parameters. Range: 0–270 Default: 15
Protein Concentration in Sample, µg/µL	The concentration of the protein in the sample. The In-Solution Digestion protocol can accommodate a wide range of concentrations, which is largely dependent on downstream needs. Range: Varies Default: 15
Concentration of Denaturant in Sample, Molar	The concentration of urea or guanidine already present in the sample. Note: The acceptable range differs for each denaturant. In addition, the value you specify influences calculations for the Denaturation Mixture. Range: 0–8 for guanidine; 0–9 for urea Default: 0.0
Total Mass per Sample, µg	Read only. The mass of the sample. The value is calculated using the Sample Volume and Protein Concentration. Range: Varies Default: 225

2	In the Reaction Setup table, type the values in the green boxes in the 2. Denaturation/Reduction area:

 

 

Field	Description
Choose denaturant from dropdown	The denaturant you want to use. Select either Guanidine or Urea.
Denaturant Concentration for this step, Molar	The concentration of guanidine or urea that you want to use in the denaturation step. To skip denaturation, enter 0. IMPORTANT If you are performing the Denaturation step, make sure the concentration is within the range for the denaturant. The permissible range ensures stability during the protocol run. Range: 0–8 for guanidine; 0–9 for urea Default: 6.00
Denaturant Concentration in Master Reagent, Molar	Read only. The concentration of guanidine or urea in the Master Reagent. To adjust the calculated value, increase or decrease the Volume of Denaturation Mixture to Add. For example, increase the Volume of Denaturation Mixture to Add to decrease the Denaturant Concentration in Master Reagent. Range: 0–8 for guanidine; 0–9 for urea Default: 9.00
Reductant Concentration for this step, Molar	The concentration of the reductant for the Denaturation step. To skip reduction, enter 0. Range: 0–0.100 Default: 0.010
Amount of Reductant, total micromoles	Read only. The micromoles of reductant. The value is calculated using the Reductant Concentration and the Denaturation Mixture volume. Range: Varies Default: 0.450
Internal Standard Stock Concentration, pmol/µL	Optional. The concentration of the internal standard stock solution you want to add to the Denaturation Mixture. Range: Varies Default: 0.0
Amount of Internal Standard in reaction, total picomoles	The total number of picomoles of the internal standard you want to include in the reaction. The value is converted to a final concentration at the indicated reaction steps (displayed in B. Reactant Concentrations Summary). Range: Varies Default: 0.0
pH Buffer (Tris) Concentration for this step, Molar	The concentration of the pH buffer present during the denaturation step. Note: Values below 0.1 M have not been tested. Values above 0.35 M are out of range for the solutions used to prepare Master Reagents. Range: 0.100–0.350 Default: 0.125
Volume of Denaturation Mixture to add, µL	The volume of Denaturation Mixture that will be added into the sample. This mixture may include denaturant, reductant, internal standard, and pH buffer as defined by the experiment. Range: 0–270 Default: 30

3	In the Reaction Setup table, type the values in the green boxes in the 3. Alkylation/Reaction area:

 

 

Field	Description
Alkylant Concentration for this step, Molar	The concentration of alkylant desired at this step. Range: Varies Default: 0.020
Amount of Alkylant, total micromoles	Read only. The number of micromoles of the alkylant. The value is calculated from the concentration and volume added to the reaction, and the final concentration at the indicated steps is displayed in the B. Reactant Concentrations Summary table. Range: Varies Default: 1.020
Volume of Alkylant to add, µL	The volume of alkylant to add. Use the default or minimum volume (5 uL) minimizes unnecessary dilution of the reaction and provides greatest flexibility in the volumes of other reactants. Range: 5–250 Default: 6.0

4	In the Reaction Setup table, type the values in the green boxes in the 4. Dilution/Alkylation Quench area:

 

 

Field	Description
Denaturant Concentration for Digest step, Molar	The concentration of guanidine or urea in the digestion step. This value determines the volume of Diluent Mixture. If the sample does not contain any denaturant, enter 0. If the Denaturant Concentration is 0, the software will allow you to manually enter a volume in the Volume of Diluent Mixture if NO Denaturant field (the last row in the 4. Dilution/Alkylation Quench area) to control the buffering capacity in the digestion reaction. Range: Varies Default: 1.00
Reductant Concentration in the Diluent Mixture, Molar	The concentration of reductant necessary for quenching the alkylant. The final concentrations of reductant and alkylant at the indicated reaction steps are summarized in the B. Reactant Concentrations Summary table. Range: Varies Default: 0.0030
Amount of Reductant added in this step, micromoles	Read only. The number of micromoles of reductant that is added during this step. The value is calculated using the Reductant Concentration and Volume of Diluent Mixture. Range: Varies Default: 0.630
Volume of Diluent Mixture to add, µL	Read only. The volume of the Diluent Mixture you want to add. The value is calculated using many of the values you supplied in the table. Range: < 250 Default: 210.0
Volume of Diluent Mixture if NO denaturant, µL	The volume of Diluent Mixture to add if Denaturant Concentration for Digest Step is 0. The Diluent Mixture contains additional reductant, if added, and pH buffer. This field appears only if you entered 0 for Denaturant Concentration for Digest Step (the first field in the 4. Dilution/Alkylation Quench area).

5	In the Reaction Setup table, type the values in the green boxes in the 5. Digestion area:

 

 

Field	Description
Protease-to-Sample Ratio (mass:mass)	The protease-to-substrate protein (sample) ratio. The default value of 50 means the final digest reaction will contain 1 µg of protease for every 50 µg of protein in the sample. Typically, this ratio varies from 1:25 to 1:100. Refer to the recommendations of the protease supplier. Range: Varies Default: 50
Protease Master Reagent Concentration, µg/µL	The concentration of the Protease Master Reagent. This value and the Protease-to-Sample Ratio determine the Volume of Protease to Add. The Protease Master Reagent should be prepared from your protease stock solution. Range: 0.05–1.50 Default: 0.500
Volume of Protease to Add, µL	Read only. The volume of Protease to add. The value is calculated using the Protease-to-Sample Ratio and the Protease Master Reagent Concentration. You can compare this value with the Volume of Digest Reaction value, below. Its lower bound is the lowest recommended volume (5 uL), and the upper bound should be no more than 10% of the Volume of Digest Reaction, for best control of reaction pH. Range: 5–50 Default: 9.0
Buffer Concentration for Digest step, Molar	Read only. The final concentration of the buffer. The value is calculated using the starting concentration and volumes added from the fields above. Make sure the value remains within the range for optimal for pH control and protease activity. Range: 0.050–0.080 Default: 0.060
Volume of Digest Reaction, µL	Read only. The digest reaction volume. The value is calculated using the volumes shown above this field. Make sure the labware you are using can accommodate this volume. The labware should also accommodate any reagents you might want to add to this volume following the overnight digestion. Range: <= 300 Default: 270

6	Review the information in the B. Reactant Concentration Summary table. If you need to make any adjustments, repeat steps 1 through 5.

 

7	Review the information in the C. In-Solution Digest form inputs table. You will transfer these values when setting up the In-Solution Digestion protocol. See Running the protocol.

 

 

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Entering values in the Reagent Prep worksheet

The Reaction Prep worksheet consists of four tables:

•	Denaturation Mixture

•

Alkylant

•

Protease

•	Diluent Mixture

Each table displays the recipe for the named reagent. Notice that the tables are prepopulated with values that have been calculated using your input in the Reaction Setup worksheet.

If you are using more denaturant than what is required, you can enter the larger mass in the Denaturation Mixer table.

The Master Reagent requires iodoacetamide powder. If you are using more than what is required, you can enter the larger mass in the Alkylant table.

 

 

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Dispensing the reagents

 

To minimize evaporation, fill the labware immediately before run time or keep the plates lidded until the run begins.

 

Before you start, make sure you have the required labware. See Labware and starter kits.

Prepare the reagents according to the recipes shown in the Reagent Prep worksheet of the Reagent Volume Calculator.

To dispense the reagents into the reservoirs:

1	Label the labware you want to use so that you can easily identify them:

•	Protease plate

•	Denaturation Mixture plate

•	Alkylant plate

•	Master Reagent plate

•	Diluent plate

2	In the Reagent Volume Calculator, do one of the following:

•	Automated Reagent Plate Setup. In the Reagent Volume Calculator, display the Automated Plate Setup worksheet, and then proceed to step 3.

•

Manual Reagent Plate Setup. In the Reagent Volume Calculator, display the Manual Plate Setup worksheet. Use manual pipettes to prepare reagent plates for the Protease, Alkylant, Denaturation, and Diluent reagents based on their respective plate layouts. Then proceed to preparing the samples (Preparing the samples) and running the protocol (Running the protocol).

3	Add the volumes of Protease, Alkylant, and Denaturation Mixture into the assigned columns of the Master Reagent plate as shown in the A. Master Reagent Plate area.

 

4	Add the volumes of the Diluent Mixture into the assigned columns of the Diluent Mixture plate as shown in the B. Diluent Mixture Plate area.

 

5	Prepare the Syringe Wash Buffer plate. Use the Single Liquid Addition v1.1 utility to fill four Greiner 96-Well Round Bottom, White Plates with the Syringe Wash Buffer.

6	If necessary, centrifuge the reagent labware to remove bubbles.

7	Run the Reagent Plate Setup protocol.

 

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Running the Reagent Plate Setup protocol

The Reagent Plate Setup protocol uses the AssayMAP Bravo Platform to transfer reagents in the Master Reagent plate to dedicated reagent plates.

To run the protocol:

1	Open the App Library. See Accessing an application.

2	Locate In-Solution Digestion: Multi-Plate, and then click App.

 

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

 

3	Click to display the Reagent Plate Setup form.

 

4	In the Application Settings area, specify the following:

 

Step	Description
Number of Sample Plates	The number of sample plates you want to process. Default: 1 Range: 1–4
Number of Columns per Plate	The number of columns in the sample plate that are occupied by samples. The value is used with the Starting Column of Reagent Plates to determine which columns in the reagent plates will contain reagents. Default: 1 Range: 1–12
Starting Column of Reagent Plates	This number depends on the first column used in the sample plates. The value in this box enables efficient use of labware. Default: 1 Range: 1–12
Protease Storage Temperature (°C)	The temperature that should be set for the Protease plate. Default: 10 Range: 4–35
Columns of Tips in Source Tip Box	The number of columns that contain pipette tips in the source tip box. If you have a partially filled tip box, make sure the empty columns are on the right side of the box. Default: 12 Range: 1–12

5	In the Step table:

a	Select the check box of the step that you want to perform. Otherwise, the protocol will skip the step during the run.

b	Enter values for the selected steps:

 

Step	Description
Add Denaturation Mixture	Specify the volume that you want added to the samples during the In-Solution Digestion run. The software automatically calculates the volume to transfer (specified volume plus overages) and then adds this volume to each well of the Denaturation Mixture plate If you skip this step, no liquid is transferred. The step is selected by default. Volume: • Default: 30* • Range: 1–250
Add Alkylant	Specify the volume that you want added to the samples during the In-Solution Digestion run. The software automatically calculates the volume to transfer (specified volume plus overages) and then adds this volume to each well of the Alkylant plate. If you skip this step, no liquid is transferred. The step is selected by default. Volume: • Default: 6* • Range: 1–250
Add Protease	Specify the volume that you want added to the samples during the In-Solution Digestion run. The software automatically calculates the volume to transfer (specified volume plus overages) and then adds this volume to each well of the Protease plate. If you skip this step, no liquid is transferred. The step is selected by default. Volume: • Default: 9* • Range: 1–250

*Use the values shown in the Reagent Volume Calculator, Reaction Setup worksheet, table C. In-Solution Digest form inputs. The total volume of Denaturation Mixture, Alkylant, Protease, Diluent Mixture, and sample must be less than 300 µL to accommodate the working volume of the U-bottom labware. The total volume should also include any liquid that will be added to the sample plate after digestion (for example, quenching by acidification).

6	Optional. Click Save Settings.

7	Confirm that the physical layout on the AssayMAP Bravo deck matches the Labware Table area and the Deck Layout area of the application.

 

Incorrect labware can cause a hardware collision, resulting in equipment damage. Ensure that the physical labware present on the Bravo deck exactly match the Labware Table and Deck Layout areas of the application.

 

The tip box at deck location 6 must contain at least three full columns of pipette tips. If you have a partially filled tip box, make sure the empty columns are on the right side of the tip box.

8	Make sure the labware are properly seated on the AssayMAP 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.

 

9	Click Run Protocol. 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.

 

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Cleaning up

When the protocol run is finished, make sure you:

•	Remove the 96AM Cartridge & Tip Seating Station from the Bravo deck and discard used pipette tips.

•	Remove the tip box and the unused pipette tips from deck location 6.

•	Remove the Master Reagent Plate from deck location 8.

•	Keep all remaining labware on the Bravo deck.

 

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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