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through which the sample is passed to separate a mixture into desired components and impurities. The result is that either the analytes of interest or the impurities in the sample are retained on the stationary phase. The other component passes through the mobile phase. Depending upon the situation, the phase that contains the desired analyte is processed for further purification. Briefly, four types of sorbent formats exist [9]: a. Cartridge: It is a small plastic or glass open-ended container filled with adsorptive particles of various types and adsorption characteristics. The cartridge type is still the most popular format b. Disc: Disk is a variation of the extraction cartridge. They consist of a 0.5 mm thick membrane where the adsorbent is immobilized in a web of micro fibrils. The sorbent (on polymer or silica) is embedded in a web of PTFE or glass fibre. c. SPE pipette tip: The solid phase sorbent is positioned inside a pipette tip, held in place by a screen and filter. The stationary phase is mixed with the sample. d. 96-well SPE plate: Each of the 96 wells has a small 1 or 2 ml SPE column with 3-10 mg of packing material. The packing material in an SPE cartridge is placed between the bottom frit or membrane and the top frit. Solid phase extraction formats are available with a variety of stationary phases, depending on the properties of protein to be isolated. Most stationary phases are based on silica that has been bonded to a specific functional group such as hydrocarbon chains (for reversed phase SPE), quaternary ammonium groups and sulfonic acid groups (for ion exchange SPE). On the basis of the sorbent used, SPE may be of following types; a. Normal phase SPE: In normal phase SPE, the stationary phase is usually composed of polar functionally bonded silica with short carbons chains. This stationary phase will adsorb polar molecules which can be collected with a more polar solvent. b. Reversed phase SPE: The stationary phase in reversed phase SPE is derivatized with long hydrocarbon chains. Less polar compounds are retained due to hydrophobic interactions. The analyte can be eluted by using a solvent with a lower polarity. c. Ion exchange SPE: It separates analytes on the basis of electrostatic interactions between the charged stationary phase and oppositely charged analytes in the sample. Ion exchange SPE may be carried out either with Cation exchange or with Anion exchange sorbents, depending upon the charge on desired analyte under given conditions. To elute the analyte, the stationary phase is washed with buffer that neutralizes the charge of either the analyte or the stationary phase. Once the charge is neutralized, the electrostatic interactions cease to exist and the analyte is eluted. A typical solid phase extraction involves four basic steps (Figure 1). First, the cartridge is equilibrated with a buffer of the same composition as the sample. The sample is then added to the cartridge. As the sample passes through the stationary phase, the desired proteins in the sample will interact and retain on the sorbent. The washing will then remove the impurities that will pass through the cartridge. Then, the protein is eluted with appropriate buffer. Figure 1: Steps in Solid phase extraction of proteins. SPE offers several advantages over traditional liquid-liquid extraction techniques, such as higher recovery and concentration of analyte, range of samples that can be used and ease of automation [9]. Chromatographic techniques Chromatography is a separation process based on distribution between two phases, a solid or liquid stationary phase and a liquid or gas mobile phase. The sample is propelled by fluid mobile phase which percolates the stationary phase. Different chromatographic processes based on characteristic principles may be used for the separation of a wide variety of substances including proteins. A given chromatographic process may be run both in low and high pressure systems, although, the basic principle remains same. Most of the protein purification procedures involve one or more of the chromatographic techniques. Based on the properties and available information about the target protein, one can choose from various available chromatography methods. OMICS Group eBooks 008
Affinity chromatography (AC): The principle of AC is the separation of proteins on the basis of a reversible interaction between the target protein and a specific ligand bound to the matrix. This protein-ligand interaction can be biological in nature or biospecific (such as Antigen-Antibody binding, Immunoglobulin-Protein A binding, Receptor-Hormone binding, Enzyme-Substrate binding, etc.) or non-biospecific (such as protein-dye binding, Histidine containing proteins-Metal ions binding). Due to its specificity, it provides a high resolution, and moderate to high capacity. The sample is applied under conditions favoring selective protein-ligand binding. Unbound material is washed out, and by applying the conditions that favor weakening of protein-ligand interaction, elution is then achieved. Elution may either be carried out specifically, using a ligand that competes with the bound ligand for binding to the target protein, or nonspecifically, by simply altering the conditions of the medium such as pH, Salt concentration, or polarity that favor the dissociation of the protein from bound ligand (Figure 2). Due to its high specificity, resolution and capacity, AC can sometimes be used for single-step purification especially when minor impurities can be tolerated. However, it is more commonly used as the first purification step. Figure 2: Scheme of Affinity chromatography A. Equilibration of affinity medium is equilibrated with binding buffer. B. Application of sample under conditions favoring reversible binding of the target molecule(s) to the ligand. The unbound material is washed out. C. Elution of the target protein by changing conditions to favor dissociation of the target protein from the ligand of the bound molecules. It can be performed specifically (using a competitive ligand), or non-specifically (by changing the pH, ionic strength or polarity). D. Re-equilibration of affinity medium with binding buffer [Adapted from ‘Affinity Chromatography, Principles and Methods’, Amersham Biosciences]. To increase the efficiency of binding of ligand to the target molecule, a spacer arm is introduced between the ligand and the matrix. This helps in avoiding the problem of steric hindrance that may occur if the target molecule binds to the ligand directly attached to the bead. Spacers are more important if the ligand is small in size. The length of a spacer arm ranges between 6-10 carbon atoms or a similar distance. A spacer must not affect the sample or the ligand or the binding between the two. Immobilization of a ligand on the matrix involves three steps. a. Activated of matrix to make it reactive toward the ligand. The matrix should be activated in a way that allows covalent attachment of a ligand with ease. b. Covalent coupling of the ligand to the matrix. c. The unreacted groups are blocked by using an excess of a suitable substance that is not supposed to interfere with the protein ligand binding (ethanolamine is one of the commonly used substance). This is to make sure that the binding occurs only at desired sites. AC simplifies the isolation process by using pre-existing, highly specific ligand-binding properties. As a result, AC is capable of giving very high purification, even from complex mixtures, in a single step. It retains its utility even when the target protein is in small amount in the mixture. Another objective that can be achieved through AC is the separation of biologically active form of the molecule from its functionally altered or its denatured form, since it is primarily based on functional properties. In addition, AC may also be employed for removal of specific contaminants. Nowadays, an increasing number of laboratory-scale purifications are performed with affinity-tagged proteins. A variety of different affinity tags, chromatography media and pre-packed columns are offered to choose optimal conditions for different proteins and purification procedures. The most common examples are the purification of Histidine-tagged proteins using Immobilized Metal Affinity Chromatography (discussed in later section) or Glutathione S-transferase-tagged proteins using immobilized glutathione. Immobilized metal ion affinity chromatography (IMAC): Immobilized Metal Affinity Chromatography (IMAC) is based on the formation of coordinate bonds between metal ions and some amino acids residues exposed on the surface of proteins (mainly histidine, but also tryptophan and cysteine). Since it is not a biospecific interaction in true sense, IMAC may be considered as a pseudo-affinity technique. The very principle of IMAC was first introduced by Porath et.al. [10]. But it has gained a deeper interest only recently due to the development of better and stable adsorbents and development of genetic engineering techniques that allow the introduction of histidine tags to proteins. The histidine tags are attached to the N- or C-terminus of recombinant proteins. Different histidine tags, from very short, such as His-Trp, to long ones, containing up to ten histidine residues, have been used. Currently, His6 tag (consisting of six consecutive histidine residues), is one of the most frequently used tags purification of recombinant proteins. The affinity of a protein for a metal depends mainly on the metal ion involved in coordination. Divalent ions of transition metals, such as, Zn 2+ , Ni 2+ , Co 2+ , Cu 2+ and Fe 2+ , are commonly used. These metal ions are immobilized on the matrix by using a suitable spacer arm plus a simple chelator which attached to the matrix . Some commonly used chelators are Imino Diacetate (IDA), Nitrotriacetic Acid (NTA) and Tris(carboxymethyl) Ethylene Diamine (TED). OMICS Group eBooks 009
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Tyrosine Nitrated Proteins: Biochem
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[58-61]. Protein sulfhydryls [62] a
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Oligoclonality of the antibody resp
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The Recent Methodologies of Protein
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Figure 6: At Domain Level, Protein
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Brownian Dynamics (BD) method Prote
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