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Team:Wageningen UR/MethodsPurification
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[[File:purification.jpg|750px|center|thumb|''Figure 1: Overview of the purification process'']] | [[File:purification.jpg|750px|center|thumb|''Figure 1: Overview of the purification process'']] | ||
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| - | The purification process consists of three parts (figure 1). The first part is differential centrifugation. It is ultracentrifugation with a sucrose gradient, this technique relies on smaller particles getting less far through the sucrose solution than larger particles [1]. The result will look like a smear on the side of the vial. We take the lowest part of the smear, in which our VLPs are. Most of the contaminants in the sample is now removed. To make it even purer, we load the sample onto an FPLC column (figure 2). FPLC or Fast Protein Liquid Chromatography is a form of liquid chromatography that is often used for purifying mixtures of proteins [2]. The FPLC can separate proteins on particle size, charge distribution (ion-exchange), hydrophobicity or reverse-phase. We used a column that | + | The purification process consists of three parts (figure 1). The first part is differential centrifugation. It is ultracentrifugation with a sucrose gradient, this technique relies on smaller particles getting less far through the sucrose solution than larger particles [1]. The result will look like a smear on the side of the vial. We take the lowest part of the smear, in which our VLPs are. Most of the contaminants in the sample is now removed. To make it even purer, we load the sample onto an FPLC column (figure 2). FPLC, or Fast Protein Liquid Chromatography, is a form of liquid chromatography that is often used for purifying mixtures of proteins [2]. The FPLC can separate proteins on particle size, charge distribution (ion-exchange), hydrophobicity or reverse-phase. We used a column that separates the particles on particle size. These kind of columns do not interact with proteins, so that our particles remain intact. |
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| - | The output of the FPLC gives us multiple fractions of a few mL. | + | The output of the FPLC gives us multiple fractions of a few mL each. These fraction contain either our VLPs, aggregates or other proteins. On a calibrated column, it is possible to predict in which fraction the VLPs are [3]. However, it can also be measured by Dynamic Light Scattering (DLS). |
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| - | [[File:FPLC.jpg|750px|center|thumb|''Figure 2: | + | [[File:FPLC.jpg|750px|center|thumb|''Figure 2: schematic of the FPLC, the solvent is taken up (1), through the degasser (2) and gradient valve (3) by the pump (5) the solvent is then taken through the loop (7) by the valve (6) to take up the sample to load it onto the column (9), the sample is run through the column and is detected by the detector (10) and is analized by the computer (11), the sample itself can be collected (12).'']] |
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| - | The complete purification methods has so far only | + | The complete purification methods has so far only been used with the wild type CCMV VLPs in our lab. Multiple variations of CCMV and HepB have been purified only by ultracentrifuge and were successful in the lab. |
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Latest revision as of 02:07, 27 September 2012
Purification of VLPs
After production, our sample still contains different types of contaminants. To remove the contaminants we build further upon the basic purification protocol that was given to us by Richard Kormelink. We changed the ultracentrifuge and the FPLC to get an even purer sample.
The purification process consists of three parts (figure 1). The first part is differential centrifugation. It is ultracentrifugation with a sucrose gradient, this technique relies on smaller particles getting less far through the sucrose solution than larger particles [1]. The result will look like a smear on the side of the vial. We take the lowest part of the smear, in which our VLPs are. Most of the contaminants in the sample is now removed. To make it even purer, we load the sample onto an FPLC column (figure 2). FPLC, or Fast Protein Liquid Chromatography, is a form of liquid chromatography that is often used for purifying mixtures of proteins [2]. The FPLC can separate proteins on particle size, charge distribution (ion-exchange), hydrophobicity or reverse-phase. We used a column that separates the particles on particle size. These kind of columns do not interact with proteins, so that our particles remain intact.
The output of the FPLC gives us multiple fractions of a few mL each. These fraction contain either our VLPs, aggregates or other proteins. On a calibrated column, it is possible to predict in which fraction the VLPs are [3]. However, it can also be measured by Dynamic Light Scattering (DLS).
The complete purification methods has so far only been used with the wild type CCMV VLPs in our lab. Multiple variations of CCMV and HepB have been purified only by ultracentrifuge and were successful in the lab.
Move further to detection of VLPs
References
- H.A. Wood, et al., Properties of viruslike particles of Penicillium chrysogenum: One double-stranded RNA molecule per particle, Virology, 1972, 7 (3), Pages 604–609
- Jie Wen, et al., Size-Exclusion Chromatography with On-Line Light-Scattering, Absorbance, and Refractive Index Detectors for Studying Proteins and Their Interactions, analytical biochemistry, 1996, 240, pages 155–166
- Julia Transfiguracion, et al., High yield purification of functional baculovirus vectors by size exclusion chromatography, J Virol Methods, 2007, 142(1-2) 21-8.
