Preparing the reagents

You can use the supplied Reagent Volume Calculator for the Fractionation application to facilitate recipe volume calculations.

 

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.

About the Reagent Volume Calculator for Fractionation

The Reagent Volume Calculator is a Microsoft Excel worksheet that:

•	Allows you to enter values such as the number of samples to process.

•	Calculates reagent needs and recipe volumes based on your input.

•	Takes into consideration dead volume, pipetting overage, and evaporation concerns.

The calculator consists of two tables:

 

Table name	Description
1. Calculator Data Entry	Allows you to enter the number of samples to be processed, the desired reagent volumes for equilibrating the cartridges or for washes, and the number of wash cycles.
2. Reagent Preparation	Uses the values you provide in the Calculator Data Entry table to calculate the volumes to use when making the reagents.

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Using the Reagent Volume Calculator

To use the Reagent Volume Calculator for Fractionation:

1	Open the App Library. See Accessing an application.

2	Locate Fractionation, and then click Calculator. 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 in the Reagent Preparation table update.

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

The fields in the Calculator Data Entry table are:

 

Field	Description
Number of Samples	The number of samples you want to process. The number of samples should be a multiple of 8. Range: 8–96 Default: 32
Columns of Cartridges Used	Read only. The number of cartridge columns and reagent columns that are required to process the samples. The value is calculated using the number of samples you specified. Range: 1–12 Default: 4
Prime — Volume	The volume of Priming Buffer used to prime the cartridges. Range: 0–250 Default: 100
Equilibration — Volume	The volume of Equilibration Buffer used to exchange the Priming Buffer in the Prime step
Cup Wash — Volume	The volume of buffer used to wash the cartridge cup and syringes to prevent sample contamination. Range: 10–100 Default: 50
Cup Wash — Wash Cycles	The number of times to wash the cartridge cup and syringes. Range: 0–10 Default: 1
Internal Cartridge Wash — Volume	The volume of Equilibration Buffer used to minimize non-target element contaminants from the cartridge resin. Range: 0–250 Default: 25
Predispense Elution Buffer — Volume	The volume of Elution Buffer to add to each fraction plate prior to collecting the fraction. This step is helpful in collecting small fraction volumes. Range: 0–50 Default: 0
Elution 1–6 — Volume	The volume of Elution Buffer for each step used to elute peptide products from the cartridges.

 

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Dispensing the reagents

 

To prevent evaporation, dispense the reagents 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 labware that are supplied with the starter kit or are approved for the application. See Starter kit, cartridges, and labware.

You will need a labware for each of the following:

•	Priming solution

•	Equilibration solution

•	Elution buffers 1–6

To dispense the reagents into the labware:

1	Label each labware according to its content.

2	Dispense the calculated volumes of reagents into the labware. The calculated volumes are in the Reagent Preparation table of the Fractionation Calculator.

Note: Fill only the columns of the labware that will be used.

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

 

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

 

 

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