For instance, epitope tags can be used as an affinity tag to perform protein purification when using stringent purification conditions or to identify interacting proteins when combining non-stringent affinity purifications and mass spectrometry analysis of co-purified proteins [ 16- 27].
As ORC has not been found previously to interact with any member of the Hat1p complex, we performed stringent reciprocal affinity tagging and purification experiments using C-terminally TAP-tagged versiofs ORCORC subunits (Orc1p-6p).
After stringent, multi-step affinity purification, mild RNase digestion, and radiolabelling, RNPs were isolated by SDS-PAGE.
Stringent selections for higher-affinity scFv variants were applied according to previously published principles, whereby the concentration of antigen was reduced over successive rounds of selection from 50 nM in round 1 down to 20pM in round 6. Screening assays were essentially performed as described.
However, our dataset from Ref. [ 16] already passed the stringent test based on socio-affinity index, taking into account the frequency of proteins within the dataset and naturally discriminating true from spurious interactions [ 16].
Using deep sequencing of expressed genes, we investigated the phylogenetic affinities of a stringent filtered set of 3,151 expressed sequence tag-contigs by generating clusters with eukaryotic homologs and constructing phylogenetic trees and networks.
The main protein copurifying with tagged Noc4p in ex vivo affinity purification experiments under stringent high salt conditions was the SSU-processome component Nop14p [28] suggesting that the Noc4p/Nop14p heterodimer represents another architectural submodule of the SSU processome.
High affinity interactions allow relatively stringent wash steps in which the majority of weakly bound proteins are removed.
To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies.
