Preparing the buffers and reagents

 

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.

Removing macromolecular particulates

Make sure the solutions are free of macromolecular particulates, such as undissolved or precipitated salts. Use a 0.22‑µm filter to filter any salt-containing solutions and minimize the possibility of clogging the AssayMAP cartridges.

 

A build-up of salts within the syringe barrels can corrode the syringe seals. Therefore, you should filter salt-containing buffers before use. In addition, you should use the System Startup/Shutdown v2.2 utility to clean the syringes after every protocol run.

 

Determining the buffer and reagent composition

This section describes buffers and reagents that may be used to strip iron from AssayMAP Fe(III)-NTA cartridges and charge them with a different metal.

•	Priming Buffer. To properly wet the surface of the resin and remove entrained air, use a priming solution containing a high amount of organic solvent. The recommended Priming Buffer is 50% ACN in water.

•	Metal Stripping Buffer. A chelator is necessary to remove the pre-charged iron from AssayMAP Fe(III)-NTA cartridges. To strip metal from the NTA cartridges, use 100 mM ethylenediaminetetraacetic acid (EDTA) prepared in high-purity water and adjusted to pH 8.0.

•

Cartridge Wash Buffer 1. This solution flushes any remaining Metal Stripping Buffer from the cartridges and conditions them with a solution that is compatible with the Metal Reagent that will be passed through the cartridge in the next step of the protocol. Good wash buffers for metal-free NTA cartridges are high-purity water or dilute acid solutions, such as 10 mM HCl, 50 mM acetic acid, or 0.1% TFA.

•

Metal Reagent. After iron has been stripped from the pre-charged Fe(III)-NTA AssayMAP cartridges, the bare NTA cartridges can be charged with different metals useful for IMAC applications. In most cases, a 100 mM solution of water-soluble metal salt prepared in high-purity water is sufficient to charge cartridges to maximum capacity (for example, 100 mM FeCl3).

Some metal solutions are highly acidic (< pH 1). However, the brief exposure of the bare NTA resin to low-pH conditions during resin charging is insufficient to degrade the performance of the cartridge using the IMAC Cartridge Customization protocol default settings.

•	Cartridge Wash Buffer 2. This solution flushes any remaining Metal Reagent from the cartridges and leaves them in a state ready for immediate use or for short-term storage. For storage guidelines, see AssayMAP cartridges.

High-purity water or a dilute acid solution, such as 10 mM HCl, 50 mM acetic acid, or 0.1% TFA is compatible with many metal-containing solutions. Cartridge Wash Buffer 2 should not cause metal precipitation, it should not form highly stable metal complexes with the immobilized metal, and it should not contain a strong chelator that could strip the immobilized metal from cartridges. Consult relevant literature for specific metals to determine chemical compatibility of selected reagents.

 

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Metal-binding capacity

The metal-binding capacity of each AssayMAP NTA cartridge is >100 nmol of Fe(III) per cartridge as determined by inductively coupled plasma—optical emission spectroscopy (ICP-OES).

 

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About the Reagent Volume Calculator for IMAC Cartridge Customization

The Reagent Volume Calculator is an Excel worksheet that you access from the Protein Sample Prep Workbench. The calculator uses formulas to calculate estimated volume requirements for buffers and reagents. Based on the number of cartridges to process, the calculator manages the dead volume, pipetting overage, and evaporation concerns for your experiment.

The following figure shows the worksheet of the IMAC Cartridge Customization Calculator.

Figure. IMAC Cartridge Customization Calculator worksheet

 

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Using the IMAC Cartridge Customization Calculator

To use the Calculator:

1	Open the App Library. See Accessing an application.

2	Locate IMAC Cartridge Customization, and then click Calculator. Microsoft Excel starts and displays the calculator.

3	Ensure that you enable content in Microsoft Excel.

4	In the calculator, enter the values in the green boxes (data entry fields):

–	Type the Number of Columns corresponding to the number of columns of AssayMAP cartridges to be used.

–	Type the volumes to use in the Prime, Strip Metal, Cup Wash 1, Internal Cartridge Wash 1, Load Metal Reagent, Cup Wash 2, and Internal Cartridge Wash 2 steps.

–	Specify the number of wash cycles used in the Cup Wash 1 and Cup Wash 2 steps.

–	Select whether or not flow-through will be collected for the applicable steps.

See the following table for a description of each field, the default values, and the range of acceptable values.

5	Select the labware that will be used. The calculator will estimate the volumes required per well, channel, or reservoir including the volume of bulk solution required to fill the labware.

Table Calculator data entry fields

 

Field	Description
Number of Columns	The number of full cartridge columns in the Cartridge & Tip Seating Station, and the corresponding number of reagent columns that will be prepared. IMPORTANT Full columns of cartridges are required to ensure proper cartridge seating on the Bravo 96AM Head. Note: One full cartridge column consists of 8 cartridges. Value: • Default: 1 • Range: 1–12
Prime	The volume (µL) of Priming Buffer at deck location 4 used to prime and properly wet the cartridge resin bed. Volume (µL): • Default: 100 • Range: 0–250
Strip Metal	The volume (µL) of Metal Stripping Buffer at deck location 5 used to chelate and remove immobilized metals from the NTA ligands of the cartridge resin bed. The Collect Flow Through option is not selected in the default protocol. If you select the Collect Flow Through option, the flow-through from this step will be collected in the Flow Through Collection plate. Volume (µL): • Default: 100 • Range: 0–250
Cup Wash 1	The volume (µL) of Cartridge Wash Buffer 1 at deck location 6 used to wash out the cartridge cup and the syringes to ensure that no Metal Stripping Buffer remains in the cup. Volume (µL): • Default: 25 • Range: 0–100
	The number of times to perform the cup wash step and its accompanying syringe wash cycle. Wash Cycles: • Default: 1 • Range: 1–10
Internal Cartridge Wash 1	The volume (µL) of Cartridge Wash Buffer 1 at deck location 6 used to flush away any remaining Metal Stripping Buffer and prepare the cartridge resin bed with buffer compatible with the Metal Reagent. The Collect Flow Through option is not selected in the default protocol. If you select the Collect Flow Through option, the flow-through from this step is collected in the Flow Through Collection plate. Volume (µL): • Default: 50 • Range: 0–250
Load Metal Reagent	The volume (µL) of Metal Reagent at deck location 9 used to immobilize metal cations onto the bare NTA ligands of the cartridge resin bed. The Collect Flow Through option is not selected in the default protocol. If you select the Collect Flow Through option, the flow-through from this step is collected in the Flow Through Collection plate. Volume (µL): • Default: 100 • Range: 0–250
Cup Wash 2	The volume (µL) of Cartridge Wash Buffer 2 at deck location 8 used to wash out the cartridge cup and the syringes to ensure that no Metal Reagent remains in the cup. Volume (µL): • Default: 25 • Range: 0–100
	The number of times to perform the cup wash step and its accompanying syringe wash cycle. Wash Cycles: • Default: 1 • Range: 1–10
Internal Cartridge Wash 2	The volume (µL) of Cartridge Wash Buffer 2 at deck location 8 used to flush away any remaining Metal Reagent and prepare the cartridge resin bed for immediate use or short-term storage. The Collect Flow Through option is not selected in the default protocol. If you select the Collect Flow Through option, the flow-through from this step is collected in the Flow Through Collection plate. Volume (µL): • Default: 50 • Range: 0–250

Note: The boxes where you can enter data provide color cues to identify how the data meets with expectations:

•	Green indicates a field where you enter a value. If the box stays green after you enter the value, the value meets all the requirements.

•	Yellow indicates a caution that the value meets only some of the requirements. Use caution in proceeding.

•	Red indicates a warning that the value does not meet the requirements.

If a box turns yellow (caution) or red (warning), flagging a potential issue, see the relevant Field description and value range in the table.

 

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Preparing the buffers and reagents

The following table lists the recommended buffers and reagents to use as a starting point for optimizing the protocol for stripping and charging Fe(III)-NTA cartridges. The simplest AssayMAP Bravo deck configuration for the default protocol uses five solutions in addition to the deionized water used for washing syringes at the wash station.

 

Buffer or reagent	Deck location and composition recommendations
Priming Buffer	Deck location 4 50% ACN : 50% H2O Organic solvent in the solution helps to purge entrained air within the cartridge resin bed and ensure the resin is properly wetted.
Metal Stripping Buffer	Deck location 5 100 mM EDTA, pH 8.0 Ethylenediaminetetraacetic acid is a strong chelator of metal cations and an aqueous solution is able to strip away metals bound to the NTA resin at a pH that is neutral to slightly basic.
Cartridge Wash Buffer 1	Deck location 6 10 mM HCl, 50 mM acetic acid, or 0.1% TFA This aqueous solution is used to flush away any remaining Metal Stripping Buffer and prepares the resin within the cartridge with a solution compatible with the Metal Reagent.
Metal Reagent	Deck location 9 50–150 mM metal salt in H2O This aqueous solution is used to immobilize metal cations to the surface of the bare NTA cartridge. Many aqueous metal solutions are highly acidic (pH < 1). Brief exposure of the resin bed to low-pH conditions during the Load Metal step will not harm the NTA resin within cartridges.
Cartridge Wash Buffer 2	Deck location 8 10 mM HCl, 50 mM acetic acid, or 0.1% TFA This aqueous solution is used to flush away any remaining Metal Reagent and prepare the cartridge resin bed for immediate use or short-term storage.

Note: All suggested solutions listed as percentages are volume/volume formulations.

 

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Setting up the labware

Plate layout requirements

Before transferring the buffers and reagents, you should plan the layout of the solutions in the microplate. Consider the following:

•	You can process 8 to 96 cartridges in parallel. Place the AssayMAP cartridges in columns of 8 cartridges in the 96AM Cartridge & Tip Seating Station. These positions must also match the locations of the buffer and reagent solutions in the microplates and reservoirs.

•	If you have fewer than 96 cartridges, make sure the cartridges occupy full columns in the 96AM Cartridge & Tip Seating Station, as the following figure shows.

The default protocol settings assume that cartridges will be arranged in multiples of 8 in a column-based configuration. Also, the Bravo Platform applies differential pressure to seat cartridges based on the number of full columns of cartridges. To achieve proper cartridge seating, entire columns must be used.

•	If the number of Fe(III)-NTA cartridges you have is not a multiple of 8, use the AssayMAP Resin-Free cartridges to fill the empty column and row positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the Cup Wash step.

Note: For a list of acceptable labware for each deck location, see AssayMAP cartridges, or see the list in the IMAC Cartridge Customization application.

Figure. Recommended microplate and reservoir layouts

 

Transferring the buffers and reagents to the microplate

Before you start, make sure you have the following:

•	The labware that are acceptable for the protocol. See Labware and deck locations.

•	The volume information from the calculator worksheet. See Using the IMAC Cartridge Customization Calculator.

An excess (overage) volume ensures that a microplate well or column does not fully deplete, which would result in aspiration of air into the syringes and then into the cartridges, compromising performance. When calculating the excess volume, also consider the evaporation of solutions on the Bravo deck. Ensure that you increase the volumes accordingly, especially for volatile organic solvents.

 

You should empirically determine the excess volume required per labware.

 

 

To minimize evaporation, prepare the buffer and reagent plates immediately before run time or keep the plates lidded until you run the protocol.

 

To transfer the buffers and reagents to the labware:

1	Label each labware to identify its contents:

•	Priming Buffer

•	Metal Stripping Buffer

•	Cartridge Wash Buffer 1

•	Cartridge Wash Buffer 2

•	Metal Reagent

2	Pipette the solutions into their appropriate labware:

a	Add the specified volume of Priming Buffer into the plate or reservoir to be placed at deck location 4. Fill only the channels or wells that will be used.

b	Add the specified volume of Metal Stripping Buffer into the plate or reservoir to be placed at deck location 5. Fill only the channels or wells that will be used.

c	Add the specified volume of Cartridge Wash Buffer 1 into the plate or reservoir to be placed at deck location 6. Fill only the channels or wells that will be used.

d	Add the specified volume of Cartridge Wash Buffer 2 into the plate or reservoir to be placed at deck location 8. Fill only the channels or wells that will be used.

e	Add the specified volume of Metal Reagent into the plate or reservoir to be placed at deck location 9. Fill only the channels or wells that will be used.

3	If necessary, centrifuge the buffer and reagent plates to remove bubbles.

 

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Labware and excess volume recommendations

The following table provides recommended volume overages of aqueous solutions for the default protocol settings. The IMAC Cartridge Customization Calculator automatically calculates the recommended excess volume for your labware selection. For details, see Using the IMAC Cartridge Customization Calculator.

Table Excess volume recommendations by labware (for aqueous solutions using the default protocol settings)

 

Labware	Mfr part number	Excess volume (µL) per well, column, or reservoir
12 Column, Low Profile Reservoir, Natural PP	Agilent 201280-100	3,000
8 Row, Low Profile Reservoir, Natural PP	Agilent 201282-100	4,500
96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom	ABgene AB-1127	30
96 Eppendorf 30129300, PCR, Full Skirt, PolyPro	Eppendorf 30129300	10
96 Greiner 652270, PCR, Full Skirt, PolyPro	Greiner 652270	10
96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt	Bio-Rad HSP‑9611	10
96 Greiner 650201 U-Bottom, Clear PolyPro	Greiner 650201	20
96 Greiner 650207_U-Bottom, White PolyPro	Greiner 650207	20
96 Greiner 651201_V-Bottom, Clear PolyPro	Greiner 651201	20
96 Costar 3363, PP Conical Bottom	Corning Costar 3363	20
96 Greiner 675801, Half Area, Flat-Bottom, UV Star	Greiner 675801	25
96 V11 Manual Fill Reservoir	Agilent G5498B#049	35,000
Reservoir, Axygen Scientific RES‑SW96-LP, 86mL, pyramid bottom	Axygen Scientific RES‑SW96-LP	20,000
Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottoms	Agilent 201254-100	20,000
Note: The recommended overages are only for aqueous solutions considering the length of time required to run the default protocol (~60 minutes). If volatile solvents are used or if the length of the protocol is increased, a larger excess volume may be required.

For details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

 

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