Before you start

This topic lists the required hardware, software, AssayMAP cartridges, labware, and reagents for running the Immobilization 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 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

The following table lists the available AssayMAP cartridges for performing Immobilization and Affinity Purification protocols 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 Protein A (PA-W) cartridge rack	G5496-60000	G5496-60018
AssayMAP Protein G (PG-W) cartridge rack	G5496-60008	–
AssayMAP Streptavidin (SA-W) cartridge rack	G5496-60010	G5496-60021
AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or as cartridge placeholders if only partial columns of Protein A, Protein G, or Streptavidin cartridges are required. For details, see Preparing the samples.	G5496-60009	–

For more details about the available cartridges, see the Agilent AssayMAP Bravo Cartridges Selection Guide.

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.

 

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Labware

Labware requirements vary depending on experimental design. The complete list of labware options and the corresponding deck locations are listed in the following table.

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	Mfr part number*	Deck location options
12 Column, Low Profile Reservoir, Natural PP	Agilent 201280-100	3, 5, 6, 7, 8
8 Row, Low Profile Reservoir, Natural PP	Agilent 201282-100	3, 5, 6, 7, 8
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 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	3, 5, 6, 8
*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. **The Greiner PCR plate is not compatible with the 25 µL cartridges at deck location 7.

 

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Reagents

The volume, type, and concentration of reagents required to immobilize antibodies or other affinity ligands onto AssayMAP cartridges for subsequent affinity purification experiments will vary depending on sample characteristics and the desired analytical result. Consult relevant literature to find specific chemistry examples for immobilizing ligands to protein A, protein G, and Streptavidin.

By default, the syringes are rinsed thoroughly with deionized water after completing the protocol to reduce the risk of premature syringe failure due to the buildup of salts within the syringe barrels. To perform more stringent syringe washing between runs, use the Syringe Wash utility. For details, see Syringe Wash v2.1.

All labware require volume overage for the protocol to execute properly. Use the Immobilization Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions.

 

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