Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris to prevent clogging the cartridges. Samples should be filtered through a 0.45‑µm filter or centrifuged at a high g‑force before loading on an AssayMAP cartridge.

The Protein Cleanup application can be used for a wide variety of proteins. Given the range of physiochemical properties that proteins display, you should examine the relevant scientific literature to determine the sample buffer conditions that favor efficient binding and avoid negatively affecting your protein of interest. In general, acidification, which can be accomplished by the addition of TFA, formic acid, or acetic acid to the sample, improves binding to reversed phase resins such as RP-W.

Agilent app note 5991-6478EN (Reference library) provides a detailed analysis of the optimal conditions for using the RP-W cartridges for the cleanup of denatured antibodies. This app note focuses on the cleanup of denatured antibodies to enable rapid trypsin digestion, but it also provides a general approach to optimize the conditions to clean up your protein of interest.

Avoid samples containing organic solvents or some types of detergents as they might inhibit binding to the cartridge. For example, loading samples in a buffer containing greater than 5% acetonitrile might inhibit binding of some proteins. If you have concerns about a specific buffer component, you should survey the scientific literature for any known effects of this type of molecule on reversed-phase resins.

The Reagent Transfer utility is a simple way to adjust the pH or dilute your sample before loading it onto the RP‑W cartridge. For instructions, see Reagent Transfer v3.0 User Guide.

The Protein Cleanup protocol permits loading of up to 1000-µL sample volume onto the AssayMAP cartridges. If the sample volume is greater than 250 µL, the protocol will iteratively load samples onto the cartridges to stay within the maximum AssayMAP syringe volume (250 µL).

Two ways to express the binding capacity of a cartridge are quantitative binding capacity and total binding capacity:

See the Agilent AssayMAP Bravo Cartridges Selection Guide for detailed information about the binding capacity for the RP-W cartridges.

If you know the approximate concentration of the target molecule in your sample and you are working within the quantitative binding range of the cartridge, you can determine the volume of sample to load as follows:

Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following:

See Labware for acceptable labware at each deck location.

An excess (overage) volume ensures that a microplate well or column does not fully deplete, which would result in aspiration of air into the syringes and then into the cartridges, compromising performance.

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. See Using the Reagent Volume Calculator for Protein Cleanup.

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 volatility of the solution and the length of the run but the recommended overages are fine for most standard runs.