Preparing the solutions

The following solutions are required for the Affinity Purification protocol:

•	Priming & Syringe Wash Buffer

•	Equilibration & Cartridge Wash Buffer

•	Elution 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 Protein Cleanup

 

 

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.

Table Protein Cleanup Calculator worksheet

 

Figure. Protein Cleanup Reagent Recipe worksheet

 

 

Return to top

Preparing the solutions

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 recommends the buffers listed in the following table as a starting point for optimizing the AssayMAP Protein Cleanup chemistry.

 

Reagent (deck location)	Composition and comments
Priming & Syringe Wash Buffer (deck location 5)	Typically 50% or greater organic (must be >25% organic), acidic, and identical in composition to the Elution Buffer to simplify the solution preparation, for example, 60% ACN: 39.9% H2O : 0.1% TFA The high-percentage organic composition is essential for reversed-phase cartridges as they ship dry and must be wetted with a high percentage organic solution or they will have very little binding capacity. This solution, the other solutions used in this application, and the sample are typically acidic. Other more mass-spec-friendly acids can substitute for TFA.
Elution Buffer (deck location 6)	High percentage organic and acidic solution, for example, 60% ACN: 39.9% H2O: 0.1% TFA Note: Carefully determine the percentage of organic solution and the pH as they both strongly influence the elution efficiency.
Equilibration & Cartridge Wash Buffer (deck location 8)	Very low or no organic and acidic solution. Typically, similar in the percentage organic and acidity to the sample, for example, 99.9% H2O : 0.1% TFA

 

Return to top

Dispensing the solutions

 

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

 

Before you start, make sure you have the following:

•	The appropriate labware for the application. See Labware.

•	The volume information from the Reagent Recipe worksheet. See Protein Cleanup Reagent Recipe worksheet.

To dispense the solutions into the labware:

1	Optional. Label each piece of labware so that you can easily identify them.

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

3	Add the specified volume of Elution Buffer into the labware to be placed at deck location 6. Fill only the channels or wells that will be used.

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

5	If necessary, centrifuge the filled labware to remove bubbles.

 

If you are using fewer than 96 cartridges, make sure you fill the labware to correspond with the sample layout in the sample plate and the cartridge positions in the 96AM Cartridge & Tip Seating Station. For more information, see Preparing the samples.

 

 

Return to top