Preparing the reagents

The following reagents are required for the On-Cartridge Reaction protocol:

•	Equilibration & Chase Buffer

•	Reagent (for example, an enzymatic or chemical agent)

•	Cartridge Wash Buffer 1

•	Cartridge Wash Buffer 2

•	Elution & Syringe Wash Buffer

To calculate the extra volume needed, use the supplied On-Cartridge Reaction Reagent Volume Calculator.

 

A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

 

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Using the Reagent Volume Calculator for On-Cartridge Reaction

 

 

The Reagent Volume Calculator is a Microsoft Excel file that contains a Calculator worksheet. You enter the number of columns to process, the volume for each step in the protocol, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration dead volume, pipetting overage, and evaporation concerns.

Figure. On-Cartridge Reaction Calculator worksheet

 

For the pipetting overage recommendations and maximum well volume for a given labware type, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

To use the Reagent Volume Calculator:

1	Open the App Library.

2	Locate the application, and then click the corresponding Calculator button. Microsoft Excel starts and displays the calculator.

3	Ensure that you enable content in Microsoft Excel.

4	Type the values in the green boxes. Notice that as you change the values in the green boxes, the calculated values change.

Note: The green box should remain green after you enter a value. If you enter a value that meets only some of the requirements, the box turns yellow. If you enter a value that does not meet any requirement, the box turns red.

To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.

Return to top

Determining the volume overage for deck location 4

 

Volume considerations for deck location 4 are very complex in this application.

 

After using the Reagent Volume Calculator to determine the volumes, make sure you take into account the following variables that can dictate the volume overage required for deck location 4:

•	Temperature of the reaction

•	Duration of the reaction

Higher temperatures and longer durations will cause more evaporation and, therefore, require more excess volume.

 

At least 3–5 µL of liquid is required in each well of a 96-well PCR plate at the end of the incubation time as this is the minimum volume required to maintain contact with the bottom of the cartridges. This contact is required to conduct heat into the resin bed. Longer incubation times or higher temperature settings require more volume, which should be determined empirically. A PCR plate is the most practical and common plate type at deck location 4 because of the low volumes of solution typically used at that location. The minimum volume required in the wells at the end of the incubation time for other plate types must be determined empirically.

 

For example, suppose you are using the Red PCR Plate Insert plus a PCR plate. To conduct a 30-minute reaction at a resin bed temperature of approximately 37 °C with a 6 µL total aspiration volume, the temperature should be set to 45 °C and 12 µL of enzyme solution should be added per well before the run is started. A volume of 12 µL is required in this example because:

•	6 µL will be aspirated.

•	3 µL are expected to evaporate.

•	3 µL are required at the end of the run to ensure heat conductance occurs during the entire run.

For more details, see the Temperature step in Assay development guidelines and protocol notes.

Return to top

Preparing the buffers and reagents

The following table describes the reagents and deck locations. The AssayMAP protocols are blind to the composition of the solutions, so you can easily adapt your optimized chemistry. Agilent Technologies recommends the buffers listed in the following table as a starting point for optimizing the AssayMAP On-Cartridge Reaction chemistry.

Note: The simplest AssayMAP Bravo deck configuration for the On-Cartridge Reaction protocol uses four to five solutions in addition to the deionized water used at the wash station.

Table Reagent preparation

 

Reagent and deck location	Composition and comments
Equilibration & Chase Buffer (deck location 3)	Should be compatible with the reagent at deck location 4. For an enzymatic On-Cartridge Reaction run, this should be a buffer optimized for the enzyme being used, which would typically be the enzyme reaction buffer. This buffer should not disrupt the affinity interaction between the cartridge resin and immobilized substrate.
Reagent (deck location 4)	Contains molecules that modify the immobilized substrate during the on-cartridge reaction. For a • Enzymatic reaction, this solution would contain the enzyme. • Chemical reaction, this solution would contain chemically active molecules.
Cartridge Wash Buffer 1 (deck location 5)	High-stringency buffer (for example, a neutral buffer with high NaCl) or a low-stringency buffer (for example, a neutral, mass-spec-compatible buffer). The AssayMAP protocol is designed to allow up to two sequential washes. The buffer selection depends on a number of factors, such as: • Whether Wash Buffer 2 will be used, which is often dependent on whether a stringent wash is desired in the protocol. If so, Wash Buffer 1 would be a stringent buffer, and Wash Buffer 2 would be a less stringent wash buffer, which would remove the stringent component of the buffer before elution. • If a stringent buffer is not used, typically, you would use Wash Buffer 1 only. The buffer would be a low-stringency wash buffer, for example, a neutral mass-spec-compatible buffer.
Cartridge Wash Buffer 2 (deck location 6)	Neutral mass-spec-compatible buffer Typically a low-stringency wash buffer. The composition of this buffer is often dictated by sensitivity of downstream steps to components of Cartridge Wash Buffer 2, as the void volume in the cartridges (~2 µL) will contain the last wash solution used before the elution step and end up in the eluate unless the Eluate Discard option is selected in the elution step and this volume is discarded.
Elution & Syringe Wash Buffer (deck location 8)	Typically a low pH solution, but this is highly dependent on the nature of the affinity interaction to be broken. A key consideration is if the sample will be neutralized following elution. If so, a weak acid that is easy to neutralize would be a good choice (for example, 12 mM HCl with 100 mM NaCl for the interaction between protein A and an antibody). However, this type of elution solution may be relatively inefficient in breaking the affinity interaction and, therefore, may require more volume for complete elution. (For example, 12 mM HCl, 100 mM NaCl requires 4–6 column volumes or 20–30 µL for complete elution of antibodies off a protein A cartridge.) If neutralization is not a key consideration or minimizing elution volume is a critical driver, a strong acid is a better choice (for example, 5% acetic acid) because complete elution can be achieved in as little as 2–3 column volumes (10–15 µL).

When preparing these reagents, you must:

•	Remove macromolecular particulates.

•	Adjust the buffer composition to optimize the pH conditions.

•	Determine the volume of solutions required using the Reagent Volume Calculator.

The prepared reagents should be kept in a closed container before use to avoid evaporation.

 

Return to top

Planning the reagent layout in the labware

The positions of the cartridges in the 96AM Cartridge & Tip Seating Station at deck location 2 dictate the positions of the solutions in labware at deck locations 3, 4, 5, 6, and 8. The following figure shows some examples.

Figure. Examples of reagent labware layout

 

For details on the cartridge layout, see Planning the cartridge layout.

See Labware requirements for acceptable labware at each deck location.

 

Return to top

Dispensing the reagents

 

To minimize evaporation, fill the labware immediately before run time or keep them covered until you run the protocol.

 

 

If you are using fewer than 96 cartridges, make sure you fill the labware columns or wells that correspond with the cartridge positions on the Cartridge & Tip Seating Station. For more information, see Planning the reagent layout in the labware.

 

You can run the Reagent Transfer utility to transfer the buffers and reagents to different labware. For instructions, see Reagent Transfer v2.1.

To dispense the reagents into the labware:

1	Optional. Label the reagent labware so that you can easily identify them.

2	Transfer the specified volume of Equilibration & Chase Buffer into the labware to be placed at deck location 3. Fill only the wells or channels that will be used.

3	Transfer the specified volume of Reagent into the labware to be placed at deck location 4. Fill only the wells or channels that will be used.

4	Transfer the specified volume of Cartridge Wash Buffer 1 into the labware to be placed at deck location 5. Fill only the wells or channels that will be used.

5	If using two wash buffers, transfer the specified volume of Cartridge Wash Buffer 2 into the labware to be placed at deck location 6. Fill only the wells or channels that will be used.

6	Transfer the specified volume of Elution & Syringe Wash Buffer into the labware to be placed at deck location 8. Fill only the wells or channels that will be used.

7	If necessary, centrifuge the reagent labware to remove bubbles.

 

Return to top