Assay development guidelines and protocol notes

This topic explains each step of the protocol so that you can optimize the In‑Solution Digestion: Multi-Plate protocol to your particular experimental design.

The first stage in the In-Solution Digestion protocol is to set up the system and washing the syringes in the Bravo 96AM Head to ensure there is no carryover from previous experiments.

Protocol stepwise guidelines

 

Protocol step	Guidelines and notes
Initial Syringe Wash	The Initial Syringe Wash step removes any 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 v1.1 utility.
Final Syringe Wash	The Final Syringe Wash step removes any 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. The amount of carryover in the syringe (expressed as dilution of the starting sample) depends on the total number of syringe wash cycles. Five cycles give a dilution of up to 105. In cases where carryover is a major concern, increasing the number of iterations also increases the dilution factor, but with a cost of increased assay time and reduced syringe lifetime.

 

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Automation movements during the protocol

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
Reagent Plate Setup	7	Checks the deck location.
	1	Washes the syringes.
	6	Presses on pipette tips.
	2	Removes pipette tips.
	6	Returns unwanted pipette tips to tip box.
	2	Picks up the pipette tips and moves them to the correct side of the 96AM Cartridge & Tip Seating Station.
	8	Aspirates the Alkylant from the Master Reagent Plate.
	7	Dispenses the Alkylant into the Alkylant plate. Repeats for the number of columns.
	2	Removes used pipette tips. Presses on clean pipette tips.
	5	Moves the Denaturation Mixture Plate and stacks it on the Alkylant Plate.
	2	Presses on pipette tips.
	8	Aspirates the Protease from the Master Reagent Plate.
	4	Dispense the Protease into the Protease Plate. Repeats for number of columns.
	2	Removes used pipette tips. Presses on clean pipette tips.
	8	Aspirates the Denaturation Mixture from the Master Reagent Plate.
	7	Dispenses the Denaturation Mixture into the Denaturation Mixture Plate. Repeats for the number of columns.
	2	Removes used pipette tips.

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
Stacking plates	2	Checks the plate.
	9	Moves Wash Plate 4 to deck location 2.
	8 6 5 3	Moves Wash Plates 3, 2, 1, and then the Protease Lid to deck location 2.

In-Solution Digestion protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the In-Solution Digestion protocol. Changing the selections or parameters will alter the movements.

 

Protocol step	Head moves to deck location...	Action
Starting protocol	2	Checks the stack.
	7	Checks the stack.
	2	Moves the Protease Lid plate to deck location 4.
Initial Syringe Wash	1	Washes the syringes.
Add Denaturation Mixture	7	Aspirates the Denaturation Mixture.
	5	Dispenses the Denaturation Mixture into sample plate 1.
	1	Washes the syringes.
	2	Moves the Syringe Wash plate from deck location 2 onto the plate at deck location 5, and performs Stringent Syringe Wash.
	1	Washes the syringes.
	Repeats the Add Denaturation moves for each sample plate. Returns all Stringent Syringe Wash plates at deck locations 9, 8, 6, and 5 to the plate stack at position 2, preserving the plate order so that the same Syringe Wash plates are used for their respective Sample plates.
Incubation	Off deck	Operator must manually move sample plates off the Bravo deck for incubation, and the manually move them back on the deck to resume the run.
Add Alkylant	7	Aspirates the Alkylant.
	5	Dispenses the Alkylant in sample plate 1 and mixes for the specified number of cycles.
	1	Washes the syringes
	2	Moves the Syringe Wash plate on to the Sample plate at deck position 5.
	5	Performs the Stringent Syringe Wash.
	1	Washes the syringes.
	Repeats the steps for sample plates at locations 6, 8, and 9, and then pauses for the on-deck Incubation for Alkylation.
	3	Moves the Denaturation Mixture plate from deck location 3 to deck location 7, uncovering the Diluent Mixture plate at deck location 3.
Add Diluent Mixture	5	Removes the Syringe Wash plate from Sample Plate 1 and returns it to the stack at deck position 2.
	3	Aspirates the Diluent Mixture.
	5	Dispenses Diluent Mixture into Sample Plate 1 and mixes for the number of cycles.
	1	Washes the syringes.
	2	Moves the Syringe Wash plate to deck position 5.
	5	Performs the Stringent Syringe Wash.
	1	Washes the syringes.
	Repeats the steps for sample plates at deck locations 6, 8, and 9.
Add Protease	5	Removes the Syringe Wash plate from Sample Plate 1 and returns it to the stack at deck location 2.
	4	Aspirates the Protease.
	5	Dispenses the Protease into Sample Plate 1 and mixes for the specified number of cycles.
	1	Washes the syringes.
	2	Moves the Syringe Wash plate to deck location 5.
	5	Performs the Stringent Syringe Wash.
	1	Washes the syringes.
	Repeats steps for sample plates in locations 6, 8, and 9. Restacks the Protease Lid and the Syringe Wash plates at deck location 2.
Final Syringe Wash	1	Washes the syringes.

 

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