Preparative Of High-quality Cytochrome C From Swine Heart Via A New Preparative Free-flow Electrophoresis Apparatus With Improvement

Target sample purification is an important step in bioanalysis for interference free and precise outcome. In the past few decades, numerous efforts have been made towards the development of innovative, simple, robust, and inexpensive separation techniques to address this issue. As a matrix-free electrophoretic technique, free-flow electrophoresis(FFE) has been applied for the separation of numerous biological samples, such as proteins, ion-pair reagents from isolated components, cells as well as organelles. Matrix-free feature of FFE make it highly unique over conventional technique, which lead to its following merits:(i) high sample recovery;(ii) good preservation of biological activity;(iii) continuous separation; and(iv) low cost.According to the size of separation chamber, there are two types of FFE, viz. large scale FFE(LS-FFE) and micro scale FFE(?-FFE). The LS-FFE has great potential for sample preparation, due to its high throughput and continuous separation. On the contrary, the ?-FFE was usually applied for sample analysis and detection owing to the merits of small sample demand and time-saving. Although LS-FFE has been developed as a separation tool for about fifty years, this approach has not been widely used for preparative separation of biological samples. This situation may be caused by the following three issues:(i) troublesome operation procedures in LS-FFE;(ii) non-ideal Joule heating dissipation in LS-FFE, which greatly reduced its repeatability; and(iii) great difficulty in developing reasonable separation methods and experimental conditions for specific biological samples.This research was consisted of three experiment parts. The parallel design of electro-conductive membrane for separation chamber to replace a ring electroconductive membrane in old design was the first part. This refreshing change is one of the highlight of this research, also brings a dramatic improvement in performance of Joule heating control--a new separation chamber was built by unconnected electroconductive membrane--to raise the upper limit of the applied voltage under good thermal control.The second part was theoptimum experiment specific to prepare Cyt C via FFE. The p H and buffer system of Carry Buffer(CB) in our improved FFE apparatus was optimized by simulating the Charge-Mass ratio(C/M) value change of three model proteins with p H of CB. Here, CLC Protein Workbench 5.0(CLC bio) software was firstly utilized to simulate the C/M value change of three model proteins with p H of CB during the procedure of FFE separation. The introduction of the software is another of the highlight of this article. The third part was thepiratical application to prepare Cyt C from nature tissue. Based on the above two parts, the high-quaility Cytochrome C(Cyt C) was prepared from the swine heart.These efforts achieved that(i) a set of improved FFE appartus was developed, it can even for commercial sales;(ii) The application field of FFE was expanded and to give some directive advice to the similar projects.(iii) A new protein preparation strategy was provided.