Preparing the samples
To minimize evaporation, prepare the samples immediately before run time or keep them covered until you run the protocol.The In-Solution Digestion application can accommodate a wide range of protein concentrations, which is largely dependent on downstream needs. Viscous samples may require dilution.
When preparing the samples, you must:
• Remove macromolecular particulates that might interfere with accurate pipetting.
• Determine the volume of samples.
• Transfer the samples to the microplate you want to use for the protocol run.
Removing macromolecular particulates
Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris.
Adjusting the sample composition
The concentration of denaturant in the sample should be entered into the In-Solution Digestion Reagent Volume Calculator. See Using the Reagent Volume Calculator for In-Solution Digestion: Multi-Plate.
Determining the volume of sample to digest
The acceptable volume of the sample is highly dependent on the digestion conditions. Use the In-Solution Digestion Reagent Volume Calculator to help determine sample volume. See Using the Reagent Volume Calculator for In-Solution Digestion: Multi-Plate.
Preparing the sample plates
Planning the microplate setup
Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following:
• You can process 8 to 96 samples per 96 well plate in parallel, and up to four sample plates. The samples should be arranged in contiguous columns, therefore, the samples should be in multiples of 8.
• The volume of samples is limited by the labware you use and the digestion reaction conditions. See Using the Reagent Volume Calculator for In-Solution Digestion: Multi-Plate. The total volume should also include any volume of liquids you intend to add post digestion.
Transferring the samples to the microplate
You can transfer the samples to the microplate that is supplied with the AssayMAP Starter Kit. See Labware and starter kits.
A small volume excess is required in all labware types to ensure proper volume transfer. The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well.
Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the solution and the length of the run but the recommended overages are fine for most standard runs.
To transfer the samples to the microplate:
1 Run the Reagent Transfer utility or Single Liquid Addition utility to transfer the samples. For instructions, see one of the following:
• If you have only one sample plate, see Reagent Transfer v3.0 User Guide.
• If you have multiple sample plates, see Single Liquid Addition v2.0 User Guide.
2 If necessary, centrifuge the sample labware to remove bubbles.