Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH₄Cl

Abstract

Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel and solutes of interest undergo repetitive precipitation-dissolution, fractionate at different locations, and elute out from the channel according to their solubility in the precipitating agent solution. We report here for the first time the use of a CPC system for fractionation of protein, RNA, and plasmid DNA in clarified lysate produced from bacterial culture. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was initially used as a precipitating agent; however, all biomolecules showed no differential solubility in the moving concentration gradient of this surfactant and, as a result, no separation of protein, RNA, and plasmid DNA occurred. To overcome this problem, inorganic salts such as NaCl and NH(4)Cl were introduced into solution of CTAB. The protein and RNA were found to have higher solubility with the addition of these salts and separated from the plasmid DNA. Decreasing surface charge density of CTAB upon addition of NaCl and NH(4)Cl was believed to lead to lower surfactant complexation, and therefore caused differential solubility and fractionation of these biomolecules. Addition of CaCl(2) did not improve solubility and separation of RNA from plasmid DNA.

Platinum group metallodrug‐protein binding studies by capillary electrophoresis – inductively coupled plasma‐mass spectrometry: A further insight into the reactivity of a novel antitumor ruthenium(III) complex toward human serum proteins

“…Since the combination of CTAB, NH 4 Cl, and NaCl is known to capable of precipitating proteins [22], the presence of ammonium in the make-up solution was considered adverse. Therefore, NH 4 NO 3 used in our prior related work [12] was replaced to a 2.5 mM phosphate buffer of pH 7.4.…”

Mathematical study on gradient formation of cetyltrimethylammonium bromide in centrifugal precipitation chromatography

“…CTAB concentration in the sample channel, on the other hand, is a strong function of the distance from the channel inlet. The concentration gradient of CTAB inside the sample channel was the gradient that facilitated the fractionation of proteins, RNA and plasmid DNA as reported by Tomanee et al (2004).…”

Centrifugal precipitation chromatography

“…In addition to the above examples, the present method has been successfully applied to many other analytes including IgG by AS gradient elution [3-5], dextran by ethanol gradient elution [9], fragments of polysaccharide by ethanol gradient elution [8], plasmid DNA, RNA and protein by the gradient elution of cationic surfactant [11, 12], and most recently carotenoid cleavage enzymes from tea leaves by ethanol gradient elution [17]. …”

“…The method has been successfully applied to various kinds of biological samples including serum proteins [1-6], IgM [2,3] and IgG [3], recombinant ketosteroid isomerase [2-5], protein-PEG (polyethylene glycol) conjugate [3, 7] polysaccharide [3, 8, 9], polymerized pigments [10] plasmid DNA RNA and protein [11, 12], etc. In this review paper, this new method is introduced by describing the principle, instrumentation, a series of basic studies and three different types of applications including the standard separation, ligand-affinity separation, and immunoaffinity separation of proteins.…”