Before you start
This topic lists the required hardware, software, AssayMAP kits, cartridges, labware, and reagents for running the Fractionation protocol. If you have questions about these items, contact Agilent Customer Service.
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. The following equipment is recommended when preparing the samples and reagents:
• Microplate centrifuge, such as the Agilent Microplate Centrifuge
• Microplate vacuum concentrator for concentration and drying of samples
• Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer
Software
The following table lists the minimum software requirements.
Software | Version |
|---|---|
Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard | 14.4 |
Agilent Protein Sample Prep Workbench | 4.0.2 |
Microsoft Excel Required for the reagent volume calculators and method setup tools. | Microsoft Office 365 32-bit edition |
For an overview of the software components, see Overview of software architecture.
Starter kit, cartridges, and labware
The starter kit for the Fractionation application contains both strong cation exchange (SCX) cartridges and labware.
Starter kit | Agilent Part number |
|---|---|
AssayMAP SCX Fractionation Starter Kit— Contains 96 strong cation exchange (SCX) cartridges and Labware for the Fractionation protocol.* | G5496-60014 |
* The SCX Fractionation kit includes16x Greiner 650201_U-Bottom, Clear PolyPro and 1x 96 ABgene 1127, 1mL Deep Well, Square Well, Round bottom plate. | |
AssayMAP cartridges
The following table lists the available AssayMAP cartridges that are commonly used to perform fractionation on the AssayMAP Bravo Platform. Each cartridge type can be purchased as a rack of 96 cartridges.
Cartridge type | Agilent part number | |
|---|---|---|
5 µL cartridge | 25 µL cartridge | |
AssayMAP SCX cartridge rack (Qty 96) | 5190-6536 | – |
AssayMAP C18 cartridge rack (Qty 96) | 5190-6532 | G5496-60017 |
AssayMAP RP-S cartridge rack (Qty 96) | G5496-60033 | G5496-60023 |
AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or as cartridge placeholders if only partial columns of SCX, C18, or RP-S 5- or 25-µL cartridges are required. For details, see Preparing the sample plates. | G5496-60009 | – |
Note: The C18 and RP-S cartridges contain reversed-phase resin. Agilent suggests using RP-S for high pH reversed-phase fractionation because it is more stable in high pH solutions.
For more details about the available cartridges, see the Agilent AssayMAP Bravo Cartridges Selection Guide or the AssayMAP Cartridges page on Agilent.com.
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 resin 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 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. Agilent does not provide a performance guarantee for cartridges that have been used more than once.
Labware
Labware requirements vary depending on experimental design. The following table provides a complete list of labware options and the corresponding deck locations.
The following figure shows the nine Bravo deck locations for labware.
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 Bravo deck (top view)
 |
Labware | Manufacturer part number* | Deck location options |
|---|---|---|
96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom | ABgene AB-1127 | 3 |
96 Eppendorf 30129300, PCR, Full Skirt, PolyPro | Eppendorf 30129300 | 4—9 |
96 Greiner 650201_U-Bottom, Clear PolyPro | Greiner 650201 | 4—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. | ||
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. For examples, consult the published scientific literature, including publications that use the AssayMAP Bravo which can be found in the Agilent AssayMAP Bravo Citation Index.
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 or increasing pH.
Similarly, both C18 and RP-S cartridges can be used for high-pH, reversed-phase fractionation using increasing concentrations of organic solvent in the elution buffers. Because of the high-pH stability of RP-S cartridges, RP-S cartridges are recommended for high-pH, reversed-phase fractionation with increasing concentrations of organic solvent.
All labware require volume overage for the protocol to execute properly. Use the Fractionation Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions.