A number of proteomics applications including, in vitrobiochemical assays, mass spectrometry, NMR and X-ray crystallography involve the use of purified proteins. Proteins may be isolated through their overexpression in organisms such as yeast, bacteria or mammalian cells in culture, or, less often, from tissue. Based on the unique characteristics of each protein, such as solubility, isoelectric point, shape, size and amino acid composition, unique approaches are developed to isolate the desired protein. The goal is to isolate as much of that functional protein as possible, with the least amount of contaminants present.
Often, the first and most important step in any protein purification procedure is centrifugation. An array of rotors available in the Avanti JXN Series (Picture 1) can be used to aid each stage of protein purification. This wide range of rotors with volume and g-force in different combinations makes the Avanti JXN a versatile instrument that can meet the requirements of any proteomics application. The instrument can be applied for different proteomics procedures such as protein concentration, density gradients, subcellular and membrane isolation, protein precipitation and cell pelleting.
Picture 1. Avanti JXN 26
Workflow for purification of overexpressed protein
Procedure for protein purification
These steps are described in more detail below:
1. Harvesting: The first step is to pellet the sample from which the protein is to be purified from such as tissues, bacterial cells, mammalian cells or insect cells. This process usually requires high-volume rotors that spin at low speed. The Avanti JXN Series includes fixed-angle JLA-8.1000/JLA-9.1000/JLA-10.500 (Picture 2) rotors for volumes of up to 6L, which can help with this. The JCF-Z continuous flow rotor is also suitable for bioprocessing setup, which demands even higher volumes of pelleting.
Picture 2. JLA-10.500 Rotor
2. Lysate clarification: Following pelleting and lysing of the cells, the next step is separation of the protein from non-protein components and cellular debris. For this purpose, high-speed clarification is used. A high-speed, low-volume rotor is required for this process. Examples of the rotors available in the Avanti JXN Series include JA-25.15, JA-25.50 and JA-30.50 Ti, which can go over 100,000 x g and therefore meet the requirements of this step.
3. Crude purification: During the precipitation process, bulk protein is recovered from a crude extract using precipitants such as polyethylene glycol and ammonium sulfate. These precipitation steps serve as primary purification methods. The precipitates are separated using rotors with a medium speed and low- to medium-volume. The JA-17, JA-18, JA-20 and JLA-16.250 rotors of the Avanti JXN Series feature an ideal combination of capacity and speed for this step.
4. Chromatography columns: The secondary purification of a target protein is usually based on a number of chromatographic principles such as size exclusion, hydrophobic, ion exchange, and affinity. Small quantities of recombinant proteins can be rapidly and easily purified using microcentrifuge spin columns and 96-well kits. These columns are suitable for high-throughput proteomics and for prescreening optimal chromatographic conditions. The JS-5.3 and JS-5.9 rotors are suitable for use with 96-well format protein prep columns.
5. Density gradient: The isolation of subcellular organelles such as plasma membranes and mitochondria from which a specific protein needs to be isolated is often achieved using density gradients. These are also used as a tertiary purification step during protein prep. The Avanti JXN Series rotors JS-24.15 and JS-24.38 can be used for both rate zonal and isopycnic density gradients.
6. Buffer exchange and concentration: It is essential to select the correct buffer and pure protein concentration for any proteomics assay. Various filter-based columns are available that can be used for buffer exchange as well as protein concentration. Several adaptors are available with the JS-4.0, JS-4.3 and JS-5.3 for these concentrators.
Picture 3. Graphical representation of volume vs centrifugal force of various Avanti JXN Series rotors used in different stages of protein purification.
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