Preparing the solutions
The following solutions are required for the IMAC Cartridge Customization protocol:
Priming Buffer
Metal Stripping Buffer
Cartridge Wash Buffers
Metal Reagent
 
*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.
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).
Using the Reagent Volume Calculator for IMAC Cartridge Customization 
 
 
The Reagent Volume Calculator is a Microsoft Excel file that contains a Calculator worksheet. You enter the number of columns to process, whether to perform the Collect Flow Through options, the volume for each step in the protocol, the number of wash cycles to conduct, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration pipetting overage and evaporation concerns.
Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.
To use the Reagent Volume Calculator:
1 Open the App Library.
2 Locate the application, and then click the corresponding Calculator button. Microsoft Excel starts and displays the calculator.
3 Ensure that you enable content in Microsoft Excel.
4 Click one of the following:
Set defaults for 5µL cartridges. Sets the values in the calculator using the values from the default method for the 5 µL cartridges.
Set defaults for 25µL cartridges. Sets the values in the calculator using the values from the default method for the 25 µL cartridges.
5 Modify the values in the green boxes as required to match your specific method. As you change the values in the green boxes, the calculated values change.
Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red.
To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.
 
 
The following figure shows the Reagent Volume Calculator.
Figure IMAC Cartridge Customization Calculator worksheet
 
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 v3.0 User Guide utility to clean the syringes after every protocol run.
 
Preparing the buffers and reagents 
The following table describes the reagents and deck locations. The AssayMAP protocols are blind to the composition of the solutions, so you can easily adapt your optimized chemistry. Agilent recommends the following buffers as a starting point for optimizing the protocol for stripping and charging Fe(III)-NTA cartridges.
Table Reagent preparation
 
Reagent (deck location)
Composition and comments
Priming Buffer
(deck location 4)
To properly wet the surface of the resin and remove entrained air, use a priming solution containing a high amount of organic solvent. Organic solvent in the solution helps to purge entrained air within the cartridge resin bed and ensure the resin is properly wetted.
The following buffer has been used successfully with the Fe(III)-NTA cartridges.
50% ACN : 50% H2O
Metal Stripping Buffer
(deck location 5)
A chelator is necessary to remove the pre-charged iron from AssayMAP Fe(III)-NTA cartridges. 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.
The following buffer has been used successfully to strip metal from the Fe(III)-NTA cartridges.
100 mM EDTA, pH 8.0
Cartridge Wash Buffer 1
(deck location 6)
This aqueous solution is used to flush away any remaining Metal Stripping Buffer and prepares the cartridge resin with a solution compatible with the Metal Reagent.
The following buffer has been used successfully with the Fe(III)-NTA cartridges.
10 mM HCl, 50 mM acetic acid, or 0.1% TFA
Metal Reagent
(deck location 9)
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.
The following reagent has been used successfully with the Fe(III)-NTA cartridges.
50–150 mM metal salt in H2O
Cartridge Wash Buffer 2
(deck location 8)
This aqueous 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.
Cartridge Wash Buffer 2 should not cause metal precipitation, form highly stable metal complexes with the immobilized metal, or 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.
The following buffer has been used successfully to load Fe(III) on the Fe(III)-NTA cartridges that have been stripped of Fe(III) using this application.
10 mM HCl, 50 mM acetic acid, or 0.1% TFA
Note: All suggested solutions listed as percentages are volume/volume formulations.
Dispensing the solutions 
 
*A small volume excess is required in all labware types to ensure proper volume transfer.
 
An excess (overage) volume ensures that a microplate well does not fully deplete, which would result in aspiration of air into the syringes and subsequently be dispensed into the resin bed, compromising performance.
The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well. See Using the Reagent Volume Calculator for IMAC Cartridge Customization.
Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the volatility of the solution and the length of the run but the recommended overages are fine for most standard runs.
 
*To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.
 
 
If you are using fewer than 96 cartridges, make sure you fill the labware to correspond with the cartridge positions in the 96AM Cartridge & Tip Seating Station. See Planning the cartridge layout.
 
To dispense the solutions into the labware:
1 Optional. Label the labware so that you can easily identify them.
2 Add the specified volume of Priming Buffer into the plate or reservoir to be placed at deck location 4.
3 Add the specified volume of Metal Stripping Buffer into the plate or reservoir to be placed at deck location 5.
4 Add the specified volume of Cartridge Wash Buffer 1 into the plate or reservoir to be placed at deck location 6.
5 Add the specified volume of Cartridge Wash Buffer 2 into the plate or reservoir to be placed at deck location 8.
6 Add the specified volume of Metal Reagent into the plate or reservoir to be placed at deck location 9.
7 If necessary, centrifuge the buffer and reagent labware to remove bubbles.
Note: You can use the Reagent Aliquot utility to dispense the buffers. For details, see Reagent Aliquot v2.0 User Guide.
See Labware for acceptable labware at each deck location.