From these 46 articles describing the scenario method in use, a total of 41 remained after a final step of exclusion (Fig. 1).
Methods that search for a single universal tree often involve steps of data exclusion in which lateral gene transfer is conceived as noise.
Through recurring steps of data exclusion, they removed as much conflicting data as possible to guarantee that no phylogenetic difference could be found between the eukaryotic and proteobacterial data.
In a hierarchical PCO analysis procedure (step by step exclusion of more distantly related samples), no increase in the cumulative percentage explained by axes 1 and 2 was observed.
The purification was done with three altering steps of size-exclusion chromatography (PD10, GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) and with spin-column centrifugation (Ultracel-50, Amicon, EMD Millipore, Billerica, MA, USA).
Briefly, synaptic vesicles were released from synaptosomes by osmotic lysis, and were further purified by rate-zonal sucrose gradient centrifugation and a final step of size exclusion chromatography on controlled pore glass beads.
A second step of size exclusion chromatography was carried out on the previous protein enriched fractions.
Mutant protein was generated using the baculovirus expression system and purified in the same way as wild-type protein (as mentioned above), with the omission of the final purification step of size exclusion chromatography, due to the relatively low expression level of the mutant proteins.
We then applied a series of exclusion steps to identify the causative mutation: (1) we removed variants that were heterozygous in the affected animal; (2) we excluded variants that were homozygous in one of the two parents; (3) we rejected variants that were inconsistent with the Mendelian inheritance model; (4) we excluded variants in the non-coding regions.
Ta0 was rapidly purified to homogeneity by a single step of size-exclusion chromatography.
