Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH(4)Cl.
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.
Tomanee
Panarat
P
Biopharmaceutical Technology Institute, Department of Chemical Engineering, Lehigh University, Bethlehem, Pennsylvania 18015, USA.
Hsu
James T
JT
Ito
Yoichiro
Y
D002498:Centrifugation; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D005591:Chemical Fractionation; D011232:Chemical Precipitation; D002845:Chromatography / Q000379:methods; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004926:Escherichia coli / Q000302:isolation & purification; D005590:Fractional Precipitation; D010957:Plasmids; D000644:Quaternary Ammonium Compounds; D012313:RNA / Q000737:chemistry*; D012965:Sodium Chloride
D002593:Cetrimonium Compounds; D004268:DNA-Binding Proteins; D000644:Quaternary Ammonium Compounds; D012965:Sodium Chloride; D012313:RNA; D004247:DNA; D000077286:Cetrimonium