Methods of assaying proteins

A method of determining characteristics of a plurality of n protein molecules, the method comprising:

• obtaining a substrate having the plurality of the n protein molecules conjugated to the substrate such that each individual protein molecule of the plurality of the n protein molecules is spatially separated from each other protein molecule of the plurality of the n protein molecules and wherein each protein molecule of the plurality of the n protein molecules has a unique, optically resolvable, spatial address;
• applying one or more fluids to the substrate, wherein the one or more fluids comprise a series of m affinity reagents, wherein each of the m affinity reagents comprises a known degree of nonspecificity and is configured to bind to one or more epitopes of at least one protein molecule of the plurality of the n protein molecules, and wherein each of the m affinity reagents is linked to an identifiable tag, wherein n is larger than m, and n and m are positive integers greater than 1, and n is at least 5 times greater than m;
• after each application of the one or more fluids to the substrate, performing the following:
• observing one or more signals of the identifiable tags linked to affinity reagents that are bound to the one or more epitopes of the individual protein molecules of the plurality of the n protein molecules;
• identifying one or more unique spatial addresses of the substrate having the one or more observed signals;
• determining that each protein molecule of the plurality of the n protein molecules having an identified unique spatial address contains the one or more epitopes having bound affinity reagents linked to identifiable tags having the one or more observed signals;
• and
• determining the characteristics of each protein molecule of the plurality of the n protein molecules containing one or more epitopes having bound affinity reagents linked to identifiable tags having the one or more observed signals using one or more deconvolution methods based on the known binding properties of the affinity reagents to match the set of affinity reagents to the protein characteristic, thereby determining the characteristic of the protein.