Preparing recombinant tau and alpha-synuclein
Tau and alpha-synuclein are intrinsically disordered, which makes them straightforward to express and unusually easy to get wrong. The preparation decides what the aggregation experiment measures, so it deserves the same attention as the assay itself.
What makes them different
Both proteins lack a stable fold, so they tolerate heat and acid that would destroy a globular protein. That property is the basis of the classic purification shortcuts: heating the lysate precipitates most host proteins while tau or synuclein stay soluble, and acid precipitation does something similar.
They also run anomalously on gels, appearing heavier than their sequence mass, which surprises people the first time and is not a sign of a problem.
Neither has a hydrophobic core to protect, so both are sensitive to their solution conditions in ways folded proteins are not, and both readily pick up modifications and adducts that change aggregation behavior.
A workable route
Expression in bacteria is standard for both. After lysis, a heat step and ion exchange will get most of the way, with a polishing step by size exclusion. Tau variants with cysteines need a decision about their redox state, because disulfide-linked dimers change aggregation markedly; many protocols reduce and then keep the protein under reducing conditions, and some use cysteine-free constructs to remove the variable entirely.
Endotoxin matters if the material will go onto cells, and bacterial expression means it has to be measured and removed.
After purification, the final buffer exchange matters more than usual. Salt, pH and the presence of a reducing agent all change the aggregation reaction, and a monomer preparation that has begun to aggregate before the experiment starts will shorten the lag phase in a way that looks like biology.
Starting from real monomer
The single most useful step before an aggregation experiment is removing pre-formed species. A size exclusion run immediately before the assay, or a high-speed spin and careful recovery of the supernatant, gives a monomer preparation with a defined starting point. Without it, the lag phase is set by whatever seeds were already there.
Confirm what you have: concentration by absorbance with a computed extinction coefficient, purity on a gel, and identity by mass if the construct is new or modified.
Making fibrils, deliberately
Tau does not aggregate readily on its own at accessible concentrations, so it is usually induced with a polyanion such as heparin, or seeded. Alpha-synuclein aggregates on shaking without an inducer, given enough time.
Both routes produce structures that differ from the ones found in disease, which matters when the material is used to raise or to test antibodies. The practice that survives review is to record the inducer, the agitation, the buffer, the temperature and the sonication step, treat the preparation as a defined reagent with a lot number, and describe it in any claim that depends on it.