Protein storage and handling
Between purification and the assay, a protein spends most of its life being handled. The handling is rarely written down, and it is a common reason two people get different answers from the same molecule.
Freeze-thaw
Each cycle stresses a protein at the freezing front, where it is concentrated, and at the ice-water interface. Some molecules tolerate many cycles; some show measurable aggregate after one. The only safe assumption is that cycles matter, so aliquot at the point of purification into single-use volumes, and record the cycle count with the sample.
Fast freezing in liquid nitrogen and fast thawing in the hand or at room temperature are usually gentler than slow freezing in a minus eighty freezer and slow thawing on ice, though this is protein-specific enough to be worth testing for anything that will be handled often.
Concentration
Concentrating on a spin filter creates a high local concentration at the membrane, which is where aggregates form. Mix during concentration rather than spinning undisturbed, and do not overshoot and dilute back; that trip through a high concentration is not free.
At the other end, very dilute protein adsorbs to plastic and disappears, which is why low-concentration standards drift over a titration series. A carrier protein or a low-binding tube fixes it when the assay tolerates one.
Buffer and pH
Aggregation depends on how far the pH sits from the isoelectric point, on ionic strength, and on specific ion effects. A panel stored in whatever buffer each purification ended in will rank by buffer. Exchange everything into one buffer before any comparison.
Reducing agents matter for proteins with free cysteines, and they expire in solution. If a construct needs a reductant, add it fresh.
Surfaces and agitation
Air-liquid interfaces unfold protein. So does vortexing, dropping a tube, and shipping on a truck. A trace of a nonionic surfactant, at the levels used in formulation, suppresses interfacial aggregation and is the standard mitigation for a molecule that has to travel or be shaken.
Light and metals
Tryptophan and methionine oxidize under light and in the presence of trace metals, particularly with peroxide from certain surfactant lots. Amber tubes and a chelator are cheap when a molecule has shown oxidation sensitivity.
What to record
Concentration, buffer, date, freeze-thaw count, and storage temperature, attached to the aliquot and carried into the assay record. When a result later looks strange, those five fields are usually enough to explain it, and their absence is usually why an investigation stalls.
A reasonable default for discovery material: purify, exchange into a defined buffer, measure concentration and monomer content, aliquot for single use, freeze fast, and re-check anything that has been stored more than a few months before it drives a decision.