Before you start
This topic lists the required hardware, software, AssayMAP cartridges, labware, and reagents for running the Phosphopeptide Enrichment 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 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 |
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
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.
AssayMAP cartridges
The following table lists the available AssayMAP cartridges for performing Phosphopeptide Enrichment 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 Fe(III)-NTA cartridge rack | G5496-60085 | – |
AssayMAP TiO2 cartridge rack | G5496-60016 | – |
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 or TiO2 cartridges, if necessary. For details, see Preparing the samples. | G5496-60009 | – |
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.
• AssayMAP TiO2 cartridges are stable from approximately pH 1 to 14.
• AssayMAP Fe(III)-NTA cartridges are stable from approximately pH 2 to 11. At levels approximately > pH 3.5, 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 approximately pH 2 to 14.
• AssayMAP Fe(III)-NTA cartridges that have been stripped (bare NTA cartridges) should be charged and used immediately.
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 |
|---|---|---|
12 Column, Low Profile Reservoir, Natural PP | Agilent 201280-100 | 3, 5–8 |
8 Row, Low Profile Reservoir, Natural PP | Agilent 201282-100 | 3, 5–8 |
96 Agilent, 2mL Square Deep Well labware | Agilent 204353-100 | 3, 7 |
96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom | ABgene AB-1127 | 3–8 |
96 Eppendorf 30129300, PCR, Full Skirt, PolyPro | Eppendorf 30129300 | 4, 7, 9 |
96 Greiner 652270, PCR, Full Skirt, PolyPro | Greiner 652270 | 4, 7, 9** |
96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt | Bio-Rad HSP‑9611 | 4, 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 | 4, 7, 9 |
96 V11 Manual Fill Reservoir | Agilent G5498B#049 | 5, 6, 8 |
Reservoir, Axygen Scientific RES-SW96-LP, 86mL pyramid bottom | Axygen Scientific RES‑SW96-LP | 5, 6, 8 |
Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottoms | Agilent 201254-100 | 5, 6, 8 |
*For dimensionally equivalent alternatives and other labware details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. **The Greiner and BioRad PCR plates are not compatible with the 25 µL cartridges at deck locations 7 and 9. | ||
Reagents
The volume, type, and concentration of reagents and buffers required for phosphopeptide enrichment will vary depending on the specific AssayMAP phosphopeptide enrichment cartridge being used, the sample characteristics, and the desired analytical result. Consult Agilent app note 5991-6073EN (Reference library) for reagent recommendations for using the AssayMAP Fe(III)-NTA with the Phosphopeptide Enrichment application. The reference library also contains references to published papers for using both Fe(III)-NTA and TiO2 for phosphopeptide enrichment. See the Agilent AssayMAP Bravo Citation Index for published papers that use the Phosphopeptide Enrichment application on the AssayMAP Bravo Platform to purify phosphopeptides from a wide range of sample matrices.
By default, the syringes are rinsed thoroughly with deionized water at the wash station after completing the protocol to reduce the risk of premature syringe failure. To perform more stringent syringe washing between runs, use the Syringe Wash utility. For details, see Syringe Wash v3.0 User Guide.
All labware require volume overage for the protocol to execute properly. Use the Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions.