Before you start

This topic lists the required hardware, software, cartridges and labware, and reagents for running the IMAC Cartridge Customization protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

Required hardware

 

 

The following figure and table show the components of the AssayMAP Bravo Platform, which is required for running the AssayMAP protocols.

Figure. AssayMAP Bravo Platform components

 

 

Item	Required hardware
1	Gripper upgrade
2	Bravo 96AM Head
3	96AM Wash Station or the later model 96 Channel Wash Station
4	Pump Module 2.0 and two carboys
5	96AM Cartridge & Tip Seating Station
6	Risers, 146 mm
7	STC controller
8	Peltier Thermal Station with custom plate nest
9	Thermal plate insert
10	Orbital Shaking Station with Control Unit

 

Optional equipment

You might need the following when preparing the reagents:

•	Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent

•	Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or equivalent

 

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Software

 

 

The following table lists the minimum software requirements:

 

Software	Version
Agilent VWorks Automation Control	13.1.5
Agilent Protein Sample Prep Workbench	3.2
Microsoft Excel Required for the reagent volume calculators and method setup tools.	2016

 

 

 

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AssayMAP cartridges

AssayMAP Fe(III)-NTA cartridges are available for performing phosphopeptide enrichment using the AssayMAP Bravo Platform. While Fe(III) is currently the most popular choice of metal cation for phosphopeptide enrichment, other metal cations such as Ga(III) and Zr(IV) have been proven effective for phosphopeptide enrichment using immobilized metal affinity chromatography (IMAC) (ref 1–6). In addition, AssayMAP Fe(III)-NTA cartridges can be functionalized with metals such as Ni(II), Co(II), Zn(II), or Cu(II) to perform IMAC purification of histidine-tagged proteins or metal-binding proteins.

The following table lists the cartridges for the IMAC Cartridge Customization application. Each cartridge type can be purchased as a rack of 96 cartridges.

 

Cartridge type	Part number
AssayMAP Fe(III)-NTA cartridge rack	G5496-60085
AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or to fill remaining positions in columns that are partially occupied by Fe(III)-NTA cartridges, if necessary. For more information, see Preparing the buffers and reagents.	G5496-60009

Cartridge use and storage guidelines

See the cartridge box label for guidelines for storing unused cartridges in the original shipping package.

Follow these guidelines to get the best performance from AssayMAP cartridges:

•	Use only particulate free buffers and reagents. Ensure that salt-containing solutions and solutions prone to precipitation are filtered through a 0.22‑µm filter.

•	Use only primed and equilibrated cartridges.

 

Cartridges ship dry and, therefore, contain air entrained in the cartridge bed. Failure to prime the cartridges can prevent the reagents and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

 

•	Do not allow wetted cartridges to dry out.

Note: Cartridges will not dry out during the course of a normal application run. Cartridges can dry out rapidly if they are exposed to air for extended periods (e.g., >1 hour) after they have been primed and equilibrated.

If you need to store primed and equilibrated cartridges for a short period, ensure that you use the lidded blue rack-receiver plate stack with an appropriate solution in the receiver plate chimneys such that the cartridge tips are submerged in the solution.

•	AssayMAP cartridges are intended to be single-use consumables.

•

AssayMAP Fe(III)-NTA cartridges are stable from pH 2 to 11, approximately. At levels > pH 3.5, approximately, resin within the cartridges may turn from pale yellow to golden yellow to orange or brown due to the formation of iron(III) complexes (most commonly with hydroxide). Resin coloration and intensity are a function of both the pH and the chemical nature of the solutions passed through the cartridge. Bare NTA cartridges are stable from pH 2 to 14, approximately.

•	AssayMAP Fe(III)-NTA cartridges that have been stripped (bare NTA cartridges) should be charged and used immediately.

Bare NTA cartridges that have been prepared properly may be stored at 4 °C to 8 °C for less than 1 week.

To prepare the cartridges for short-term storage, flush the cartridges with 20% ethanol and place them in the 96AM Cartridge Rack and Receiver Plate with 150 µL of 20% ethanol in each well.

 

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Labware and deck locations

The following table lists all labware options for the protocol and the deck locations at which the labware are permitted. The accompanying figure shows the labware locations on the AssayMAP Bravo deck.

 

Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

 

Figure. Labware locations on the AssayMAP Bravo deck (top view)

 

 

Labware	Mfr part number*	Deck location options
12 Column, Low Profile Reservoir, Natural PP	Agilent 201280-100	3—9
8 Row, Low Profile Reservoir, Natural PP	Agilent 201282-100	3—9
96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom	ABgene AB-1127	3—8
96 Eppendorf 30129300, PCR, Full Skirt, PolyPro	Eppendorf 30129300	7, 9
96 Greiner 652270, PCR, Full Skirt, PolyPro	Greiner 652270	7, 9
96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt	Bio-Rad HSP‑9611	7, 9
96 Greiner 650201 U-Bottom, Clear PolyPro	Greiner 650201	3—9
96 Greiner 650207_U-Bottom, White PolyPro	Greiner 650207	3—9
96 Greiner 651201_V-Bottom, Clear PolyPro	Greiner 651201	3—9
96 Costar 3363, PP Conical Bottom	Corning Costar 3363	3—9
96 Greiner 675801, Half Area, Flat-Bottom, UV Star	Greiner 675801	7, 9
96 V11 Manual Fill Reservoir	Agilent G5498B#049	4, 5, 6, 8, 9
Reservoir, Axygen Scientific RES‑SW96-LP, 86mL, pyramid bottom	Axygen Scientific RES‑SW96-LP	4, 5, 6, 8, 9
Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottoms	Agilent 201254-100	4, 5, 6, 8, 9
*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

 

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Buffers and reagents

The typical deck configuration for the IMAC Cartridge Customization protocol has the following:

•	Priming buffer

•	Metal-stripping buffer

•	Two internal cartridge wash buffers

•	Metal-containing reagent

•	Purified deionized water (H2O) for the wash station

Always use high-purity reagents for all solutions.

 

Make sure you filter salt-containing buffers if salt precipitation is a risk. Salt precipitates can clog cartridges and cause poor performance.

 

 

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 Shutdown utility to clean the syringes after every protocol run.

 

 

Use the Reagent Volume Calculator for IMAC Cartridge Customization to determine volumes and suggested overages for the selected labware and specific protocol conditions. See Preparing the buffers and reagents.

 

 

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