Preparing the solutions

The following solutions are required for the Affinity Purification protocol:

•	Priming & Equilibration Buffer

•	Cartridge Wash Buffers

•	Elution & Stringent Syringe Wash Buffer

 

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 Affinity Purification

 

 

The Reagent Volume Calculator is a Microsoft Excel file that contains the following:

•	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 pipetting overage and evaporation concerns.

•	Reagent Recipe worksheet. You enter the concentrations of each component in your reagent, and the worksheet calculates the recipe volumes required.

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 is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red.

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

 

 

The following figures show the worksheets of the Reagent Volume Calculator.

Figure. Affinity Purification Calculator worksheet

 

Figure. Affinity Purification Reagent Recipe worksheet

 

 

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Preparing the buffers

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 following buffers as a starting point for optimizing the AssayMAP affinity purification chemistry.

Table Reagent preparation

 

Reagent (deck location)	Composition and comments
Priming & Equilibration Buffer (deck location 3)	Typically a buffered aqueous solution with neutral pH and physiologic salt concentration, such as Phosphate-Buffered Saline (PBS), that is similar in composition to the buffer solution used to prepare the sample.
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, PBS or a neutral mass-spec-friendly buffer) The Affinity Purification application is designed to allow the use of two wash buffers for high and low stringency washes or a single wash buffer. The buffer selection depends on a number of factors. First, consider whether Cartridge Wash Buffer 2 will or will not be used. This decision is often dependent on whether or not a stringent wash is desired in the protocol. If so, one would typically use the stringent buffer for Cartridge Wash Buffer 1 and then a less stringent wash buffer for Cartridge Wash Buffer 2, which would remove the stringent component of the buffer before elution. If a stringent buffer is not used, you would typically use Cartridge Wash Buffer 1 only, and it would be a low-stringency wash buffer, such as PBS or a neutral mass-spec-friendly buffer.
Cartridge Wash Buffer 2 (deck location 6)	Typically, a low-stringency buffer (for example PBS or a neutral mass-spec-friendly buffer) The composition of this buffer is often dictated by sensitivity of downstream steps to components of Wash Buffer 2 as the void volume in the cartridges (~2 µL for 5 µL cartridges or 10 µL for 25 µL cartridges) will contain the last wash solution used before the Eluate step and end up in the eluate unless the Discard Eluate option is selected and this volume is discarded.
Elution & Stringent Syringe Wash Buffer (deck location 8)	12 mM HCl with 100 mM NaCl pH 2.0 or 5% acetic acid The elution solution is typically a low pH solution. A key consideration is if the sample will be neutralized following elution. If so, a 12 mM HCl solution with 100 mM NaCl is a good choice as it is very easy to neutralize. However, this solution requires approximately 4—6 column volumes to elute antibodies off the cartridge. If neutralization is not a key consideration or minimizing elution volume is a critical driver, a 5% acetic acid solution is a good choice because targets can be eluted in as little as 2 column volumes.

 

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Dispensing the solutions

 

To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

 

To dispense the solutions into the labware:

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

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

3	Add 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.

4	If using two wash buffers, add 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.

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

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

 

If you are using fewer than 96 cartridges, make sure you fill the labware to correspond with the samples at deck location 4 and cartridge positions on the Cartridge & Tip Seating Station. For more information, see Preparing the samples.

 

 

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