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Protein Purification Protein Purification

Copyright 2006 Molecular Station Copyright 2006 Molekylær Station

Protein purification is vital in the characterisation of your protein of interest. Protein rensning er afgørende i den karakterisering af dine proteiner af interesse. Purification of your protein allows one to study the function of the protein, and its enzymatic activity. Rensning af din protein tillader en at studere den funktion af proteinet, og dets enzymatiske aktivitet. Stuctural information from the protein can also be obtained from purified proteins including NMR, 3-D information such as protein crystallization. Stuctural oplysninger fra protein kan også være fremstillet af rensede proteiner, herunder NMR, 3-D oplysninger såsom protein crystallization.

So how does one go about purifying a protein? Så hvordan gør man rensende et protein?

Proteins can be purified according to size, solubility, Charge and Binding affinity Proteiner kan blive renset efter størrelse, opløselighed, Charge og bindende affinitet

Proteins can readily be visualized and differentiated by electrophoresis methods.  Theses gel techniques can also be used to obtain small quantities (micrograms) of purified polypeptides.  However, they do not provide large amounts of purified proteins in their native state.  Substantial quantities of purified proteins, of the order of many milligrams, are needed to elucidate fully their three-dimensional structure and their mechanism of action.  Several thousand proteins have been purified in active form on the basis of such characteristics as size, solubility, charge and specific binding affinity.  At each step in purification, the preparation is assayed for a distinctive property of the protein of interest (eg enzymatic activity) to assess the efficacy of the procedure. Proteiner umiddelbart kan visualiseres og differentieret ved elektroforese metoder. Theses gel teknik kan også bruges til at få små mængder (mikrogram) af renset polypeptider. Men de giver ikke store mængder af rensede proteiner i deres oprindelige tilstand. Betydelige mængder af renset proteiner , I størrelsesordenen mange milligram, der er nødvendige for at belyse fuldt ud deres tre-dimensionelle struktur og deres mekanisme for handling. Flere tusinde proteiner er blevet renset i aktiv form på grundlag af sådanne egenskaber som størrelse, opløselighed, oplade og specifikke bindende tilhørsforhold. På hvert trin i rensning, præparatet er analyseret for en karakteristisk egenskab af protein af interesse (f.eks enzymatiske aktivitet) til at vurdere effekten af proceduren.

Proteins can be separated from small molecules by dialysis through a semi-permeable membrane, such as cellulose membrane with pores.  Molecules having dimensions significantly greater than the pore diameter are retained inside the dialysis bag, wwhereas smaller molecules and ions traverse the pores of such a membrane and emerge in the dialysis outside the bag. Proteiner kan adskilles fra små molekyler ved dialyse gennem et semi-permeable membran, såsom cellulose membranen med porer. Molekyler, der har dimensioner betydeligt større end den porediameter er bevaret inde i dialyse taske, wwhereas små molekyler og ioner krydse porer af en sådan membranen og dukker op i dialyse uden for posen.

More discriminating separation on the basis of size can be achieved by the technique of gel-filtration chromatography.  The sample is applied to the top of the column consisting of porous beads made of an insoluble but highly hydrated polymer such as dextran or agarose (which are carbohydrates) or polyacrylamide.  Sephadex, Sepharose, and Bio-gel are commonly used commercial preparations of these beads, which are typically 100 micrometers in diameter.  Small molecules can enter these beads but large ones cannot.  The result is that small molecules are distributed both in the aqueous solution inside the beads and between them, whereas large molecules are located only in the solution between the beads.  Large molecules flow more rapidly through this column and emerge first, because a smaller volume is accessible to them.  It should be noted that the order of emergence of molecules from a column of porous beads is the reverse of the order in gel electrophoresis, in which a continuous polymer framework impedes the movement of large molecules.  Much larger quantities of protein can be separated by gel filtration chromatography than by gel electrophoresis but at the price of lower resolution. Mere diskriminere adskillelse på grundlag af deres størrelse kan opnås ved teknikken gel-filtrering kromatografi. Prøven er anvendt til toppen af kolonne bestående af porøse perler lavet af en uopløselig, men meget hydreret polymer såsom dextran eller agarose (som er kulhydrater) eller polyacrylamid. Sephadex, Sepharose, og Bio-gel er almindeligt anvendt kommercielle præparater af disse perler, som typisk 100 mikrometer i diameter. Små molekyler kan indtaste disse perler, men de store ikke kan. Resultatet er, at små molekyler er fordelt både i vandig opløsning inde i perler og mellem dem, mens store molekyler er placeret kun i opløsningen mellem perler. store molekyler flow hurtigere gennem denne kolonne og emerge første, fordi en mindre mængde er tilgængeligt for dem. Det skal bemærkes, at rækkefølgen af fremkomsten af molekyler fra en kolonne med porøst perler er det omvendte af den rækkefølge, gelelektroforese, hvor en kontinuerlig polymer rammer hæmmer den frie bevægelighed for store molekyler. Meget større mængder af proteiner kan adskilles ved gel filtrering kromatografi end med gel elektroforese, men på bekostning af lavere opløsning.

The solubility of most proteins is lowered at high salt concentrations.  This effect, called salting out, is very useful, though not well understood.  The dependence of solubility on salt concentration 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. Opløseligheden af de fleste proteiner er sænket ved høje saltkoncentrationer. Denne effekt, kaldet saltning, er meget nyttig, men ikke godt. Afhængighed opløselighed på salt koncentration adskiller sig fra det ene protein til en anden. Derfor saltning ud kan bruges til at fractionate proteiner. Saltning out er også nyttigt for at koncentrere fortyndede opløsninger af proteiner.

Copyright 2006 Molecular Station Copyright 2006 Molekylær Station