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Introduction
Modes of liquid chromatography are generally divided in accordance with their aims, principles, and technology used for chromatography. Three methods are generally used in protein analysis as the most precise and reliable: affinity method, SDS-page method, and size-exclusion chromatography method. These methods are used for purifying protein solvents by tagging the necessary sample, and then infiltrating the solvents using one of the three offered methods.
Affinity Method
This method is based on using the clearly defined biochemical principle of non-covalent interaction between proteins and solvent that is performed on the selective nature with components that is generally observed between specific and analyte molecules. As it is stated by Lehninger and Cox (207), this method is specific enough, however, this can not be regarded as robust, and the key purpose of this method is to arrange a protein purification process with non-tagged proteins.
However, proteins are tagged with particular composites like His-tags, or biotin. When the purification is over, these tags are removed for clearing the solvent, and leaving pure protein in the test tubes. In accordance with the research by Sahin and Tetaud (20), affinity chromatography is often used with using metal compounds and molecules for the procedure:
An IMAC system using EBA technology (Streamline chelating)was performed with the Streamline 25 column containing 90 ml of Streamline chelating gel corresponding to a sedimented bed height of 19 cm. The Streamline 25 column was linked to a Biopilot workstation. The evaluation of bed stability was performed by visual inspection, and by measuring the degree of expansion according to the manufacturers instructions.
SDS Page Method
This method is based on the principle that the given columns of protein molecules may be insufficient for purifying, or extracting the necessary analytes. Therefore, this method involves directing a series of unresolved peaks with varying psycho-chemical properties. (Gaberc-Porekar, and Menart, 337) Considering the fact that the actual retention mechanism differs from the initial dimensional separation, the compounds may be separated by using different dimensions. Hence, the sample is located on a plate, purified, dried, and then it is rotated by 90 degrees for redeveloping in a second solvent. As it is stated by Klepsch and Schlegel (48):
In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDSPAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDSPAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis.
Therefore, membranes of the proteins help researchers in the purification procedure. Two-dimensional principle of protein purification is often used when the analytes can not be separated from the solvent with affinity or any other method. Therefore, SDS method is more reliable, and offers better results, however, it requires thorough preparation for the procedure, as well as it is labor intensive.
Size Exclusion
This may be also called gel permeation chromatography, as gels are often used as the basis of the purification solvents. Protein solution are used in denaturing conditions, and the technique presupposes exclusion of the proteins of a particular size. Hence, smaller molecules travel through a porous matrix. Hence, this may be used only for molecules of one size. Often, there is a need to purify proteins of various sizes, hence, a variable volume of eluents used for the purification. Eluent is pooled in different tubes, and each contains are precise quantity of the protein that should be purified. In accordance with the research by Kunji, Harding and Butler (62):
Size exclusion chromatography is an established technique for the determination of hydrodynamic volumes of proteins or protein complexes. When applied to membrane proteins, the contribution of the detergent micelle, which is required to keep the protein soluble in the aqueous phase, needs to be determined to obtain accurate measurements for the protein. In a detergent series, in which the detergents differ only by the length of the alkyl chain, the contribution of the detergent micelle to the hydrodynamic volume is variable, whereas the contribution of the protein is constant.
In the light of this statement, it should be emphasized that this is one of the most universal and reliable method, considering the labor intense, as well as the opportunity of purifying solvents without proper preparation for the procedure.
His-Tagged Recombinant Protein
The results of his-tagged purification of protein solvents depend on the method used for this procedure. Hence, the actual importance of defining the necessary samples is crucial for the results and applying the purification matrix (if the purification is performed by size exclusion method). Tagging is useful for using the solvents of various natures, and, it should be stated that the entire process of purification can not deal without properly tagged proteins that are used either as the samples, or as the locators of the similar molecules of proteins.
Works Cited
Gaberc-Porekar, Vladka; Menart, Viktor. Perspectives of Immobilized-Metal Affinity Chromatography. Journal of Biochemichal and Biophysical Methods 49 (2001) 335360.
Klepsch, Mirjam; Schlegel, Susan. Immobilization of the first Dimension in 2D blue Native/SDSPAGE Allows the Relative Quantification of Membrane Proteomes. Methods 46 (2008) 4853.
Kunji, Edmund; Harding, Marilyn; Butler, Jonathan. Determination of the Molecular Mass and Dimensions of Membrane Proteins by Size Exclusion Chromatography. Methods 46 (2008) 6272.
Lehninger. Alan; Cox, Michael 1993. Principles of Biochemistry, USA, Worth Publisher Inc.
Sahin, Annelise; Tetaud, Emmanuel. LdARL-1 His-tagged Recombinant Protein: Purification by Immobilized Metal Affinity Expanded Bed Adsorption. Journal of Chromatography. B, 818 (2005) 1922.
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