Before you start

This topic lists the required hardware, software, AssayMAP kits, labware, and reagents for running the Fractionation 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 are 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

 

To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 µL cartridges.

 

Note: The white wide-bore chimneys work for both 5-µL and 25-µL cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide.

 

 

Optional equipment

You might need the following when preparing the samples and reagents:

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

•	Microplate vacuum concentrator for concentration and drying of samples

•	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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Starter kit, cartridges, and labware

The following table displays the starter kit that is available for the Fractionation application. The starter kit contains both strong cation exchange (SCX) cartridges and labware.

Note: There is no Fractionation starter kit that contains C18 or reversed-phase (RP-S) cartridges. To order C18 or RP-S cartridges, see AssayMAP cartridges.

 

Kit	Part number
AssayMAP SCX Fractionation Starter Kit— Contains 96 strong cation exchange (SCX) cartridges and Labware for the Fractionation protocol	G5496-60014

AssayMAP cartridges

You can purchase each of the following cartridge types as an individual rack of 96 cartridges without the labware. To order the cartridges, contact Automation Solutions Customer Service.

Note: The SCX cartridges are included in the starter kit.

Note: This application has not yet been optimized for the 25 µL cartridges.

 

Cartridge type	Part number
AssayMAP SCX cartridge rack (Qty 96)	5190-6536
AssayMAP C18 cartridge rack (Qty 96)	5190-6532
AssayMAP RP-S cartridge rack (Qty 96)	G5496-60033
AssayMAP Resin-Free cartridge rack (Qty 96) This cartridge can be used for mock runs or as cartridge placeholders if only partial columns of SCX, C18, or RP-S cartridges are required. For details, see Transferring the samples to labware.	G5496-60009

Cartridge use and storage guidelines

See the cartridge box label for storage guidelines.

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

•	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.

•	PA-W, SA-W, and PG-W cartridges tolerate brief exposure to pH as low as 2.0. The stability of the PA-W, SA-W, and PG-W cartridges after capturing additional affinity ligands should be determined empirically.

Labware

 

Use only the labware supplied with the starter kit and place them at the AssayMAP Bravo deck locations specified in the instructions. Using other labware or placing labware at unapproved deck locations can cause crashes during protocol runs and damage hardware components.

 

The following labware are required for the Fractionation application. They are included in the starter kit. However, you can also purchase them separately.

 

Labware	Part number*
96-Well Round-Bottom, Clear Plates (Qty up to 16, or 2 x number of fractions + 4)	Greiner 650201
1.2-mL Deep-Well Plates (Qty 1)	ABgene AB-1127
*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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Reagents

The AssayMAP Fractionation protocol is designed to permit multiple types of fractionation to be performed. Therefore, the reagent requirements are dependent on the fractionation strategy and the chemistry of the AssayMAP cartridges.

In general, buffer systems used for liquid chromatography separations in a column format can be adapted for use in the AssayMAP cartridge format. Fractionation using AssayMAP SCX cartridges can be accomplished using elution buffers with increasing ionic strength. Alternatively, fractionation can be achieved using elution buffers with increasing pH and SCX cartridges.

Similarly, both C18 and RP-S cartridges can be used to for low-pH, reversed-phase fractionation using increasing concentrations of organic solvent in the elution buffers. Because of the high-pH stability of RP-S cartridges, they may be used to achieve high-pH, reversed-phase fractionation with increasing concentrations of organic solvent.

 

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