Assay development guidelines and protocol notes

This topic explain each step of the protocol so that you can optimize the affinity purification protocol to your particular experimental design.

Protocol stepwise guidelines

 

Protocol step	Guidelines or notes
Number of Full Columns of Cartridges	This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct. 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 damage both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 10 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required. • Less than the actual number of columns 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 due to liquid moving past the syringe cartridges seal into the cartridge cup. Default: 1 Range: 1-12
Initial Syringe Wash	The Initial Syringe Wash step uses the wash station to flush any potential contaminants from the syringes in the head. 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 to waste. Wash Cycles: • Default: 3 • Range: 0–10
Prime Cartridges	The syringes aspirate the specified volume of Priming Buffer to perform the Prime Cartridges step. After mounting the cartridges on the probes, the head moves to the Organic Waste plate to prime the cartridges by dispensing Priming Buffer through the cartridges to waste. Cartridges are parked at the seating station and the probe head moves the syringes to the wash station at deck location 1 and washes the syringes with 250 µL of water for the specified number of cycles. A rate slower than 300 µL/min will likely have no benefit and may prevent complete removal of air. A rate faster than 300 µL/min may not sufficiently penetrate the pores in the resin preventing the entire cartridge bed from being properly wetted. Volume: • Default: 100 • Range: 0–250 Flow Rate: • Default: 300 • Range: 0.5–300 Wash Cycles: • Default: 1 • Range: 0–10
Equilibrate Cartridges	The Equilibrate Cartridges step exchanges the buffer used for priming with equilibration buffer within the resin bed. It is critical to equilibrate the cartridge bed with a buffer that establishes optimal chemical conditions for analyte binding. In the default protocol, this step is selected. The syringes aspirate the specified volume of Equilibration Buffer to perform the Equilibrate Cartridges step and then dispenses at the specified flow rate through the cartridges into the Organic Waste plate. The head moves to the seating station and parks the cartridges. The head moves the syringes to the wash station at deck location 1 and washes the syringes with 250 µL of water for the specified number of cycles. Volume: • Default: 50 • Range: 0–250 Flow Rate: • Default: 10 • Range: 0.5–300 Wash Cycles: • Default: 1 • Range: 0–10
Load Sample	During Load Sample, the Bravo 96AM Head aspirates a user-defined volume into the syringes, mounts the cartridges onto the syringes, dispenses the specified volume of samples through the cartridges at the specified flow rate into Flow Through Collection at deck location 8 (Collect Flow Through step enabled) or Organic Waste at deck location 3, and performs an external cartridge tip wash at the wash station (where cartridge tips dip into the wash station chimneys to remove any sample on the outside of the cartridge). Finally, the head moves the syringes to the wash station at deck location 1 and washes the syringes with 250 µL of water for the specified number of cycles. The volume depends on the concentration and the mass of sample to capture. It is not possible to aspirate all of the liquid from a microplate well. Add at least 15-20 µL extra sample liquid to each well to ensure that the sample does not deplete fully, which would result in aspiration of air into the cartridges negatively impacting performance. Binding capacity is inversely proportional to flow rate. For many resins, a flow rate of 20 µL/min results in a low dynamic binding capacity. It also produces some loss of total binding capacity, as the sample does not have sufficient time to diffuse throughout the pores of the resin in the cartridge bed. If the flow rate is too slow, the protocol can run at non-optimal speed without significant additional binding. Generally, the syringes should be washed as part of the Load Sample step. Under certain circumstances, you can skip the wash step to minimize run time. Volume: • Default: 100 • Range: 0–250 Flow Rate: • Default: 5 • Range: 0.5–300 Wash Cycles: • Default: 3 • Range: 0–10
Collect Flow Through	Selecting the Collect Flow Through step enables the sample flow-through to be directly eluted into the Flow Through Collection plate at deck location 8 during the Load Sample step. If the Load Sample step is conducted, but the Collect Flow Through step is not selected, the sample flow-through will be dispensed directly into the Organic Waste plate at deck location 3. The Collect Flow Through step is skipped in the protocol if the Load Sample step is not conducted.
Cup Wash	The Cup Wash addresses the small amount of sample liquid that may remain above the upper cartridge membrane after sample loading. This step removes the residual liquid by filling the cartridge cups with utility buffer and then removing it along with the residual sample solution. You control the number of Cup Wash cycles by changing the number of wash cycles in the application. Each Cup Wash is followed by a syringe wash at the wash station. Volume: • Default: 50 • Range: 10–100 Wash Cycles: • Default: 1 • Range: 0–10
Internal Cartridge Wash	The Bravo 96AM aspirates a user-defined volume of equilibration buffer into the syringes, mounts the cartridges, and dispenses that volume at the defined flow rate through the cartridge. The head parks the cartridges at the Tip Seating Station at deck location 2 and then washes syringes at the wash station with 250 µL of deionized water. Volume: • Default: 25 • Range: 0–250 Flow Rate: • Default: 5 • Range: 0.5–300 Wash Cycles: • Default: 3 • Range: 0–10
Collect Flow Through	Selecting the Add Wash to Flow Through step enables the volume of liquid used for the Internal Cartridge Wash to be directly eluted into the Flow Through Collection plate at deck location 8 during the Internal Cartridge Wash step. If the Internal Cartridge Wash step is conducted, but the Add Wash to Flow Through step is not selected, the flow-through from the wash will be dispensed directly into the Organic Waste plate at deck location 3. The Add Wash to Flow Through step is skipped in the protocol if the Internal Cartridge Wash step is not conducted.
Predispense Elution Buffer	The Predispense Elution Buffer step transfers a specified volume of Elution Buffer from the appropriate Elution Buffer plate (1-6) to the corresponding Fraction Collection plate before eluting each fraction into the Fraction Collection plate. Predispensing Elution Buffer into a Fraction Collection plate maximizes the recovery of small volume elutions (< ~ 20 µL) by allowing direct elution into a liquid. Small volumes may cling to the end of the cartridge during elution that cannot be removed by the programmed tip touches in the well of the plates. • Default: 15 • Range: 0–50
Elute Fraction 1–6	Before running the Fractionation protocol, the Stack Plates protocol stacked a set of Elution Buffer plates at deck location 4 and a set of Fraction Collection plates at deck location 7. The number of Fraction Collections plates is equal to the Number of Fractions you specified. During this step, the plate movements on the deck are as follows: • The Sample plate (deck location 5) and Flow Through Collection plate (deck location 8) move one position to the right and are stacked on the Priming Buffer plate and Equilibration Buffer plate at deck locations 6 and 9, respectively. • To collect Fractions 1-4, the Elution Buffer and Fraction Collection plates are moved from the stacks in positions 4 and 7 to the central deck positions (5 and 8) to perform the fraction collection. Then, once fraction collection is complete, they are stacked at the far right deck positions (6 and 9). For example: the Elution Buffer plate 1 is moved from the stack of Elution Buffer plates at location 4 to location 5. Fraction Collection plate 1 is moved from the stack of Fraction Collection plates at location 7 to location 8. Elution buffer is aspirated into the syringes from Elution Buffer Plate 1 at location 5. If the Predispense Elution Buffer step was selected, this volume will be dispensed to Fraction Collection plate 1 at location 8. Cartridges are mounted and samples are eluted from the cartridge directly into the Fraction Collection plate 1. Cartridges are parked at deck location 2 and an optional syringe wash is conducted at the wash station. Fraction Collection plate 1 is moved from location 8 and stacked on location 9. Elution Buffer plate 1 is moved from location 5 and stacked on location 6. • Steps 2 and 3 are repeated for fractions 2, 3, and 4. • For fraction 5, Step 2 is conducted but instead of stacking the Elution Buffer and Fraction Collection plates (step 3), they remain at locations 5 and 8, respectively. • For fraction 6, there is no plate movement. Elution Buffer plate 6 remains at location 4 and Fraction Collection plate 6 remains at location 7. Final Fraction Collection plates 1-4 will be in deck location 9, Fraction Collection plate 5 will be in deck location 8 and Fraction Collection plate 6 will be in deck location 7. Volume: • Default: 25 • Range: 0–250 Flow Rate: • Default: 5 • Range: 0.5–300 Wash Cycles: • Default: 1 • Range: 0–10
Final Syringe Wash	The Final Syringe Wash uses deionized water from the wash station to flush any potential contaminants from the syringes. The head performs these washes on the wash station. The AssayMAP Bravo aspirates 250 µL of fresh deionized water into the syringes using the bare probes and dispenses at an offset between the chimneys on the wash station. Increase the number of Wash Cycles to better clean the syringes. Increasing the Wash Cycles value increases the duration of the assay and consumption of reagents, and affects wear on the syringes. Wash Cycles: Default: 3 Range: 0–10

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Automation movements during the protocol

Stack Plates protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Stack Plates protocol. Changing the Number of Fractions will alter the movements.

 

Protocol step	Head moves to deck location...	Action
Stack Plates	4	Checks the labware height.
	6	Checks the labware height.
	8	Moves Elution Buffer plate 1 to deck location 4.
	5 9 6 3	Moves remaining Elution Buffer plates to deck location 4, in the order listed.
	8	Moves Fraction Collection plate 1 to deck location 7.
	5 9 6 3	Moves remaining Fraction Collection plates to deck location 7, in the order listed.

Fractionation protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Fractionation protocol. Changing the selections or parameters will alter the movements.

 

Protocol step	Head moves to deck location...	Action
Starting protocol	4	Checks the Elution Buffer plate stack height.
	7	Checks Fraction Collection plate stack height.
	9	Checks labware height.
	6	Checks labware height.
	2	Parks all cartridges that might have been loaded on the head from a previously aborted protocol.
Initial Syringe Wash	1	Washes the syringes.
Prime Cartridges	6	Aspirates the Priming Buffer.
	2	Mounts the cartridges.
	3	Dispense the Priming Buffer through the cartridges and into Organic Waste Plate.
	2	Park the cartridges.
	1	Wash the syringes.
Equilibrate Cartridges	9	Aspirates the Equilibration Buffer.
	2	Mounts the cartridges.
	3	Dispense the Equilibration Buffer through the cartridges into the Organic Waste plate.
	2	Parks the cartridges.
	1	Washes the syringes, if applicable.
Load Samples	5	Aspirates the samples.
	2	Load the cartridges.
Collection Flow Through	8	Loads samples onto cartridges, collects flow-through.
	1	Washes the cartridge exteriors.
	2	Parks the cartridges.
	1	Washes the syringes.
Cup Wash	9	Aspirates the Equilibration Buffer.
	2	Performs Cup Wash.
	3	Dispenses the buffer into the Organic Waste plate.
	1	Washes the syringes.
Internal Cartridge Wash	9	Aspirates the Equilibration Buffer.
	2	Mounts cartridges.
	8/5	Dispenses the wash into Flow Through Collection or Organic Waste.
	1	Washes the cartridge exteriors.
	2	Parks the cartridges.
	1	Washes the syringes.
	5	Moves the Sample plate to deck location 6.
	8	Moves the Flow Through Collection plate to deck location 9.
Elute Fractions 1–6	4	Moves Elution Buffer plate 1 to deck location 5.
	7	Moves Fraction Collection plate 1 to deck location 8
	5	Aspirates the Elution buffer for predispense and elution.
	7	Dispenses the predispense volume into the Fraction Collection plate.
	2	Mounts the cartridges.
	7	Elutes into Fraction Collection plate 1.
	1	Washes the cartridge exteriors.
	2	Parks the cartridges.
	1	Washes the syringes.
	8	Moves Fraction Collection plate 1 to deck location 9.
	5	Moves Elution Buffer plate 1 to deck location 6.
	Repeats for fractions 2, 3, and 4.
	Repeats for fraction 5, except the final plates remain at deck locations 5 and 8.
	Repeats for fraction 6, except the plates remain at deck locations 4 and 7.
Final Syringe Wash	1	Wash the syringes.
	Final Fraction Collection plates 1-4 will be in deck location 9, Fraction Collection plate 5 will be in deck location 8, and Fraction Collection plate 7 will be in deck location 7.
	1	Wash the syringes.

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