Protein Purification

protein purification why do we need purified protein by analyzing pure protein we can determine amino acid sequences and investigate the proteins biochemical function besides crystallization of proteins need pure protein and we can obtain x-ray data from the protein crystals with a picture of tertiary structure which is the actual functional unit of the protein for pure protein is so important for further analysis we will discuss the protein purification methods which usually used purified protein contains only one type of protein which we are interested in at beginning this protein may be only a tiny fraction of the complex how could we know we isolate the particular protein we need we need an assay for some unique identifying property of the protein so that we can tell when the protein is present or whether this is a target protein determining an effective assay is often difficult for enzymes which are protein catalysts the assay is usually based on the reaction that the enzyme catalyzes in the cell for already known protein there is a more convenient method Western blot we can use specific antibody to analyze the target protein these methods will be used after the purification process we'll talk about this later when you know how to analyze whether your purification process is successful we can start to purify the protein now first the protein need be released from cell there are several methods to disrupt the cell membrane sonication is a common method others include French press enzymatic or chemical cleavage of the cells and freeze sign for larger-scale continuous disruption high pressure homogenization or bead milling are widely used after that step the mixture is fractionated by centrifugation here we need make some trials or dependent on previous experience to make sure the target protein enriched in which component of the fractions the differential centrifugation yields the dense pellet of heavy material at the bottom of the centrifuge tube and a lighter supernatant above then the supernatant is again centrifuge that a greater force to yield yet another pellet and supernatant in the Centrify Gatien process the mixture yields several fractions of decreasing density each still containing hundreds of different proteins which are subsequently a sage for the further purification proteins could be purified according to their basic characteristics such as soul ability size charge and specific binding affinity usually protein mixtures are subjected to a series of separations each based on a different property to yield a pure protein most proteins are less soluble at high salt concentrations an effect called salting out the salt concentration at which a protein precipitates differs from one protein to another hence salting out can be used to fractionate proteins salting out is also useful for concentrating dilute solutions of proteins including active fractions obtained from other purification steps dialysis can be used to remove the salt and above step proteins can be separated from small molecules by dialysis through a semipermeable membrane such as a cellulose membrane with pores proteins of greater dimensions than the pore diameter and are retained inside the dialysis whereas smaller molecules and ions Traverse outside the bag through the pore however this process could not distinguish between proteins effectively chromatography is the more discriminating separations on the basis of size the column consists of porous beads made of insoluble but highly hydrated polymers such as dextran or accuracy or poly acrylamide a we applied sample on the top of a column the small molecules can enter the porous of the beads but large ones cannot thus the small molecule goes both inside and between the beads whereas large one goes only between the beads small molecules flow longer distance than the larger molecules so the large molecules flow more rapidly through this column and emerge first molecules that are of a size to occasionally enter a bead will flow from the column at an intermediate position and small molecules which take a longer tortuous path will exit last I an exchange chromatography proteins may have net positive or negative charge at different pH range this is another basic rules for proteins separate if a protein has a net positive charge it will usually bind to a column of beads containing carboxylate groups whereas a negatively charged protein will bind to a column of beads containing diethyl amine oasl group protein sample loads on the column a positively charged protein binds to the beads and other charged protein will flow through this column and emerge first the positively charged protein for example can been eluded by increasing the concentration of sodium chloride or another salt in the eluding buffer because sodium ions compete with positively charged groups on the protein for binding to the column proteins with less net positive charge released first then the higher charge density proteins there are lots of ion exchange chromatography you need to choose suitable chromatography depends on the character of your interest in protein affinity chromatography affinity chromatography is another powerful and generally applicable means of purifying proteins this technique takes advantage of the high affinity of many proteins for specific chemical groups for example nee polystyrene resin is widely used for the purification of his tag protein his protein has affinity for any polystyrene when passing a column containing the polystyrene his tied protein attached to the modified resin whereas most other proteins do not affinity chromatography is a powerful means of isolating transcription factors a protein mixture is percolated through a column containing specific DNA sequences attached to a matrix proteins with a high affinity for the sequence will bind and be retained after that the transcription factor could be washed by a high concentration salt solution in conclusion affinity chromatography isolate target protein in three steps firstly attaching the chemical group which could affinity with target protein to the column secondly loading the sample into the column and let the target protein affinity with chemical group thirdly eluding the target protein by a high concentration salt solution or other method to decrease binding affinity high pressure liquid chromatography the resolving power of all of the column techniques can be improved substantially through the use of a technique called high pressure liquid chromatography this techniques could enhance the ability of other columns discussed above the column materials themselves are much more finely divided and as a consequence there are more interactions sites and thus greater resolving power when using HPLC the samples load into the column and move through the stationary phase under the pressure for different material have different affinity with stationary phase the objects leave the column in different time the detector obtains various peak signal and each peak represents a different kind of compound after comparing analysis of these signals we will know the material contained in the sample the net result is high resolution as well as rapid separation after all the purification steps how could we know our purification scheme is effective apart from specific activity rises in each purification step another method is using electrophoresis to display the target proteins at each step proteins can be separated largely on the basis of mass by electrophoresis in a polyacrylamide gel under denaturing conditions SDS polyacrylamide gel electrophoresis is rapid sensitive and capable of a high degree of resolution we can examine the efficacy of our purification scheme by analyzing a part of each fraction by SDS page the initial fractions will display dozens to hundreds of proteins as the purification progresses the number of bands will diminish and the prominence of one of the bands should increase this band will correspond to the protein of interest in case some protein may have similar weight with the target protein we need to run isoelectric focusing first isoelectric focusing can be combined with SDS page to obtain very high resolution separations proteins can also be separated electro phoretic li on the basis of their relative contents of acidic and basic residues the isoelectric point of a protein is the pH at which its net charge is zero protein with similar weight are less possible have same pie thus using two dimensional electrophoresis proteins have been separated in the horizontal direction on the basis of isoelectric point and in the vertical direction on the basis of mass besides quality analysis of protein purification we also need quantify the target protein at each step if the target protein is an enzyme we could determine the protein concentration by enzyme activity on the other hand if the target protein doesn't have the enzyme activity we could semi quantify the target protein through SDS page gel which could analysis the percentage of the target protein and total sample a good purification scheme takes into account both purification levels and yields a high degree of purification and a poor yield leave little protein with which to experiment a high yield with low purification leaves many contaminants in the fraction and complicates the interpretation of experiments finally mass spectrometry could determine the target protein mass more precisely these methods are called matrix assisted laser desorption ionization and electrospray spectrometry using this method we could learn more about our target protein we provide protein expression and purification services in our company if you have any questions on protein purification welcome to contact us for details our website is www.martialartsnc.com

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Keywords: protein, protein purification, protein purify, purification method, chromatography

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Length: 11min 43sec (703 seconds)

Published: Thu Sep 14 2017