Basic Principles used in the Selection of Monobeads™ion Exchangers for the Separation of Biopolymers

Ion‐Exchange Chromatography

“…For those ion exchanger types which allow rapid separations, optimal starting pH and choice of anion or cation exchanger can be determined using chromatographic titration curves (18).…”

Low-tech electrophoresis, small but beautiful, and effective: Electrophoretic titration curves of proteins

“…The theoretical considerations outlined above have been applied to planning the purification protocols for creatinine kinase [76], carbonic anhydrase [77], urinary proteins [78], and L-toxin of Clostridium sordellii [16]. The concepts above have also been stressed in a visually impressive way by comparing densitometry of the ETC pattern and ion exchange elution profile at the same pH (urinary proteins [78]).…”

Determining the Identity and Structure of Recombinant Proteins

“…Protein titration curves (in reality, pH/mobility curves) became suddenly popular in the late seventies after an original report by Rosengren et al (), who demonstrated a simple method to force a protein to migrate across a quasi‐stationary pH gradient in such a way as to trace a sigmoidal path resembling a classical potentiometric titration curve. These pH/mobility curves were found to be quite useful in a number of applications: (1) for screening of genetic mutants (Righetti et al, ; Righetti et al, ); (2) for studying macromolecule‐ligand interactions (Krishnamoorthy et al, ; Constans et al, ); (3) for demonstrating macromolecule‐macromolecule interactions (Righetti et al, ; Lostanlen et al, ; Gianazza et al, ); (4) for direct pK determination of simple cations and anions and of uni‐uni‐valent amphoteric molecules (Righetti et al, ; Valentini et al, ); (5) for determining dissociation constants ( K d ) of ligands and proteins and their pH dependence (Ek et al, ; Ek and Righetti, ); (6) for detecting protein and peptide microheterogeneity (Arnaud et al, ; Henner and Sitrin, ; Picard et al, ; Van Den Oetelaar and Hoenders, ); and (7) for choosing optimal pH conditions in ion‐exchange chromatography (Fägerstam et al, ).…”

“…Determining the Identity and Structure of Recombinant Proteins determination of simple cations and anions and of uni-uni-valent amphoteric molecules (Righetti et al, 1979;Valentini et al, 1980); (5) for determining dissociation constants (K d ) of ligands and proteins and their pH dependence ; (6) for detecting protein and peptide microheterogeneity (Arnaud et al, 1980;Henner and Sitrin, 1984;Picard et al, 1987;Van Den Oetelaar and Hoenders, 1987); and (7) for choosing optimal pH conditions in ion-exchange chromatography (Fägerstam et al, 1983). The times must have been ripe for such an idea, because at just about the same time, Creighton (1979) reported a method for studying the unfolding of proteins by running them across a urea gradient: these denaturation curves, often sigmoidal in shape, represent the transition from the folded to the unfolded state.…”