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Principles for fractionation of protein mixtures using centrifugal ultrafiltration devices
Author(s) -
Koticha Darshan,
Kulkarni Subodh,
Chaudhry Pulkit,
Chhabra Shrina,
Thanaseelan Ramya,
Brewster David,
Sharma Ajay,
Lynch John
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a634
Subject(s) - fractionation , ultrafiltration (renal) , chromatography , chemistry , diafiltration , size exclusion chromatography , yield (engineering) , regenerated cellulose , membrane , fraction (chemistry) , cellulose , materials science , microfiltration , biochemistry , metallurgy , enzyme
Fractionation is the process of separating a mixture into its components using a combination of physico‐chemical properties of the solute. We demonstrate efficient fractionation of protein solutions on the basis of molecular size, using centrifugal ultrafiltration devices with an Ultracel regenerated cellulose membrane. Using these devices, cytochrome C (12 kDa) was effectively purified from a mixture containing 10% serum. A model for fractionation was generated using 12 protein pairs in a designed experimental plan. Yields and purity of the proteins were measured spectroscopically and by SDS‐PAGE. Our results show that devices with Ultracel membranes are critical for efficient fractionation. The optimal centrifugal force for fractionation is uniform across all molecular weight cutoffs (MWCO). Fractionation efficiency is improved with multiple diafiltration cycles. For optimal yield (>50%) and purity (>90%), the ratio of the molecular weights of the proteins and the ratio of the MWCO to the size of the passing solute should be >/= 2. Moreover, high starting concentration of retained, but not passing solute, may affect the fractionation yield. In conclusion, this work describes a simple approach for protein fractionation using ultrafiltration devices. This approach offers several advantages for fractionation over traditional gel filtration, including increased sample size, speed and ease‐of‐use.