Protein Sample Prep Workbench : In‑Solution Digestion: Multi-Plate v2.0 User Guide : Assay development guidelines and protocol notes
Assay development guidelines and protocol notes
This topic explains the following:
• Each step of the protocol so that you can optimize the Digestion protocol to your particular experimental design
• Automation movements during the protocols
For details on how to use the Experiments Editor, see Using Experiment IDs.
Protocol stepwise guidelines 
 
Protocol step
Guidelines and notes
Initial Syringe Wash
The Initial Syringe Wash step removes potential contaminants from the syringes. During each Initial Syringe Wash cycle, the head aspirates 250 µL into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste.
You can increase the number of wash cycles to better clean the syringes. However, be aware that increasing the number of wash cycles also increases protocol run time and reduced syringe lifespan.
Add Denaturation Mixture
The Add Denaturation Mixture step prepares the reaction for denaturation and may include denaturant, reductant, internal standard, and buffer based on user-specified experimental design. The Denaturation Mixture is made during the Reagent Plate Setup protocol as the components are automatically transferred from the Master Plate into the Denaturation Mixture plate.
Incubation (denaturation- off deck)
The denaturation incubation step is used when incubation for denaturation or reduction is required.
The step pauses the protocol run so that you can manually move the sample plates off the Bravo deck, manually seal the plates using the PlateLoc Sealer or equivalent device, and incubate the plates for the desired length of time.
After the incubation period, remove the seals from the plates, place the plates back on the Bravo deck, and then resume the protocol run.
Add Alkylant
Depending on the specific reagent used for alkylation, the reaction might be light sensitive. To protect the alkylation reaction from light, the Bravo Platform covers the sample plates with wash plates for the duration of the alkylant incubation step.
Incubation (Alkylation on deck)
The Incubation for alkylation step incubates the lidded samples on the Bravo deck. At the end of the incubation period, the wash plates are removed from the sample plates.
Add Diluent Mixture
The Diluent Mixture prepares the reaction for digestion by diluting the denaturant in the sample with buffer to a concentration in which the protease is active and optionally adding a reductant to quench unreacted alkylant.
Add Protease
The In-Solution Digestion protocol is designed to accommodate any proteolytic enzyme. You can add more enzyme using the Single Liquid Addition utility. See Single Liquid Addition v2.0 User Guide.
Final Syringe Wash
The Final Syringe Wash step removes potential contaminants from the syringes. During each Final Syringe Wash cycle, the head aspirates 250 µL of DI water into the syringes using the bare probes, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste.
In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions.
Automation movements during Reagent Plate Setup protocol 
This section describes the basic movements of the AssayMAP Bravo Platform during the Reagent Plate Setup protocol.
 
Protocol step
Head moves to deck location...
Action
Start protocol
NA
Sets the Peltier Thermal Station to the Protease Storage Temperature (°C) specified in the form.
7
Checks the plate height.
1
Dispenses any liquid remaining in the syringes.
Performs 3 up-and-down cycles of the syringes above the wash station to dry the syringes.
Prepares pipette tips
6
Mounts all the pipette tips present in the tip box.
Note: The pipette tips are arranged in the tip box in full columns starting at column 1.
2
Parks the pipette tips in the seating station.
2
Mounts all the pipette tips in the seating station, except columns 1–3, with a head offset so that the pipette tips in column 4 are mounted onto column 1 (left most column) of the head.
6
Returns unwanted pipette tips to tip box.
2
Mounts the pipette tips in column 1–3 of the seating station and moves them to columns 10–12 of the seating station.
Alkylant solution transfer
2
Mounts the pipette tips from seating station column 12 onto column 1 of the head.
8
Prewets the pipette tips with Alkylant solution.
8
Aspirates the Alkylant solution, including a small excess.
7
Dispenses the Alkylant solution.
8&7
Repeats the preceding two steps as necessary to complete filling the wells.
8
Dispenses the excess Alkylant solution back into the Alkylant solution.
2
Ejects the used pipette tips into column 1 of the seating station.
Protease solution transfer
5
Picks up the Denaturation Mixture Plate.
7
Puts the Denaturation Mixture Plate on the Alkylant Plate.
2
Mounts the pipette tips from column 11 of the seating station onto column 1 of the head.
8
Prewets the pipette tips with Protease solution.
8
Aspirates the Protease solution, including a small excess.
4
Dispenses the Protease solution.
8&4
Repeats the preceding two steps as necessary to complete filling the wells.
8
Dispenses the excess Protease solution back into the Protease solution.
2
Ejects the used pipette tips into column 2 of the seating station.
Denaturation Mixture transfer
2
Mounts the pipette tips from column 10 of the seating station onto column 1 of the head.
8
Prewets the pipette tips with Denaturation Mixture.
8
Aspirates the Denaturation Mixture, including a small excess.
7
Dispenses the Denaturation Mixture.
8&7
Repeats the preceding two steps as necessary to complete filling the wells.
8
Dispenses the excess Denaturation Mixture back into the Denaturation Mixture.
2
Ejects the used pipette tips into column 3 of the seating station.
Automation movements during Plate Stacking protocol  
This section describes the basic movements of the AssayMAP Bravo Platform during the Plate Stacking protocol.
 
Protocol step
Head moves to deck location...
Action
Start protocol
2
Checks the stack height.
Stack plates
9
Moves Syringe Wash plate 4 to deck location 2.
8
6
5
3
Moves Syringe Wash plates 3, 2, 1, and then the Protease Lid to deck location 2.
Automation movements during Digestion protocol 
This section describes the basic movements of the AssayMAP Bravo Platform during the Digestion protocol. Changing the selections or parameters will alter the movements.
 
Protocol step
Head moves to deck location...
Action
Start protocol
–
Sets the Peltier Thermal Station to the temperature specified on the form.
2
Checks the stack height.
7
Checks the stack height.
2
Picks up the Protease Lid plate.
4
Puts the Protease Lid plate on the Protease plate.
Initial Syringe Wash
1
Washes the syringes
Add Denaturation Mixture
7
Aspirates the Denaturation Mixture.
5
Dispenses the Denaturation Mixture, and then mixes the sample.
1
Washes the syringes.
2
Picks up a Syringe Wash plate from the top of the stack.
5
Puts the Syringe Wash plate on top of the Sample plate, and then performs a Stringent Syringe Wash.
1
Washes the syringes.
For each remaining Sample plate:
Repeats the Add Denaturation moves starting with aspirating Denaturation Mixture from deck location 7.
Returns all Stringent Syringe Wash plates at deck locations 9, 8, 6, and 5 to the plate stack at deck location 2, preserving the plate order so that the same Syringe Wash plates are used for their respective Sample plates.
Incubation
Off deck
Protocol pauses and incubation message appears.
Operator must manually move Sample plates off deck for incubation, and then move them back on deck and click Continue to resume the run.
Add Alkylant
7
Picks up the Denaturation Mixture plate.
3
Puts the Denaturation Mixture plate on top of the Diluent plate.
1
Pauses to wait for temperature to reach set point +/- 5 °C.
7
Aspirates the Alkylant.
5
Dispenses the Alkylant and mixes the sample.
1
Washes the syringes.
2
Picks up a Syringe Wash plate from the wash plate stack.
5
Puts the Syringe Wash plate on top of the Sample plate, and then performs a Stringent Syringe Wash.
1
Washes the syringes.
For each remaining Sample plate:
Repeats the Add Alkylant moves starting with aspirating Alkylant from deck location 7.
3
Picks up the Denaturation Mixture plate.
7
Puts the Denaturation Mixture plate on top of the Alkylant plate.
Add Diluent Mixture
5
Picks up the Syringe Wash plate.
2
Puts the Syringe Wash plate on top of the stack.
3
Aspirates the Diluent Mixture.
5
Dispenses the Diluent Mixture and mixes the sample.
1
Washes the syringes.
2
Picks up a Syringe Wash plate from the top of the stack.
5
Puts the Syringe Wash plate on the Sample plate, and then performs a Stringent Syringe Wash.
1
Washes the syringes.
For each remaining Sample plate:
Repeats the Add Diluent Mixture moves starting with picking up the Syringe Wash plate.
Add Protease
4
Picks up the Protease Lid plate.
2
Puts the Protease Lid plate on top of the stack.
Picks up Protease Lid plate.
3
Puts the Protease Lid plate on top of the Diluent plate.
5
Picks up the Syringe Wash plate.
2
Puts the Syringe Wash plate on top of the stack.
3
Aspirates the Protease.
5
Dispenses the Protease and mixes the sample.
1
Washes the syringes.
2
Picks up a Syringe Wash plate from the wash plate stack.
5
Puts the Syringe Wash plate on top of the Sample plate, and then performs a Stringent Syringe Wash.
1
Washes the syringes.
For each remaining Sample plate:
Repeats the Add Protease moves, except the Protease aspiration happens before picking up of the Syringe Wash plate from the Sample plate and placing it on the stack at deck location 2.
Returns all Syringe Wash plates at deck locations 9, 8, 6, and 5 to the plate stack at deck location 2, preserving the plate order.
3
Picks up the Protease Lid plate.
2
Puts the Protease Lid plate on top of the stack.
Final Syringe Wash
1
Washes the syringes.