For the co-purification of interaction partners with NirF, the STrEP-tagged (C-terminal) version of NirF was produced in the P. aeruginosa PAO1 strain RM301 lacking the native NirF.
The robust methods of expression, purification and measurement of interactions in vitro that we report here for RPSA and its domains, should allow one to assay additional ligands or inhibitors, and identify precisely their binding sites by mutagenesis.
We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA.
Carboxylic acid group and defect sites may be formed on SWNT sidewalls as a result of purification steps, and interaction with CO molecules likely occurred [34].
However, it should be noted that conventional biochemical and affinity purification approaches are prone to false-positive identification of interaction partners.
We attempted to obtain further insight into the biological function of Fun30 by identification of interaction partners using TAP (Tandem Affinity Purification) of endogenously tagged Fun30, but did not find any interaction partner using this approach (data not shown).
Further purification of extracted LTA was by hydrophobic interaction chromatography as described [35], using a HiTrap™ Octyl Sepharose FF 1 ml column (Pharmacia).
(A ) Affinity purification of the Cik1 Kar3 complex from different cell cycle states and identification of interaction partners by mass spectrometry.
This article also aims to sketch prospects for further development of the use of protein protein interactions for the purification of proteins.
Here, we established a novel method, engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP), for purification of specific genomic regions retaining molecular interactions.
