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 optimal chemical environment for binding is generally similar for protein A, protein G, and streptavidin.

One of the most important considerations for optimizing binding conditions is the pH of the sample, which should be near neutral pH. Both low (2 to 3) and high (10 to 11) pH ranges can prevent binding to protein A or protein G resins. The sample should generally be:

Protein A, protein G, and streptavidin generally tolerate moderate levels of salt, non-ionic detergents, and mild denaturing reagents, such as urea, quite well. You should examine scientific literature for the known effects and tolerances of protein A, protein G, and streptavidin, keeping in mind that these tolerances may differ depending on the antibody species and subtype for protein A and protein G.

Protein A and protein G bind a wide variety of antibody subtypes and species.1 Carefully consider the species and subtype of antibody when choosing between using an AssayMAP Protein A or Protein G cartridge for purification.

The antibodies that bind to protein G largely overlap the set that binds to protein A. While protein A is the industry standard for purification and titer determination of human therapeutic antibodies, protein G is the standard for purification of antibodies used as bioanalytical tools, primarily because many antibody subtypes are generated in species that bind poorly to protein A, for example, mouse IgG1 and rat IgG1.2

The AssayMAP Immobilization protocol permits loading up to 1000 µL of sample onto AssayMAP cartridges. For sample volumes > 250 µL, the protocol will iteratively load samples onto cartridges to stay within the maximum syringe volume (250 µL) of the Bravo 96AM Head.

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 Protein A, Protein G, and Streptavidin 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:

The amount of affinity ligand to immobilize depends on the amount of target. Use at least a 5‑fold molar excess compared to the amount of target to be captured. A low molar excess of affinity ligand (approximately 5-fold) requires a very slow loading flow rate (approximately 2 µL/min). As the molar excess increases so too can the loading flow rate. The exact molar excess and the flow rate should be determined empirical as they depend on many factors such as the strength of the affinity interaction and the diffusion rate of the target molecule.

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 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.

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.