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Protein Purification Proèi¹æavanje proteina

Copyright 2006 Molecular Station Copyright 2006 Molekularna kolodvor

Protein purification is vital in the characterisation of your protein of interest. Protein purification je bitno u karakterizacija proteina Va¹eg interesa. Purification of your protein allows one to study the function of the protein, and its enzymatic activity. Proèi¹æavanje Va¹e proteina omoguæuje da studija funkcija proteina, a njeni enzimska aktivnost. Stuctural information from the protein can also be obtained from purified proteins including NMR, 3-D information such as protein crystallization. Stuctural informacije iz proteina takoðer se mogu dobiti iz oèistili proteina, ukljuèujuæi NMR, 3-D informacije kao ¹to je protein crystallization.

So how does one go about purifying a protein? Tako je kako se jednom go o èi¹æenju jedan protein?

Proteins can be purified according to size, solubility, Charge and Binding affinity Proteini mogu biti proèi¹æena prema velièini, topljivost, Napunite i obvezujuæa afinitet

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. Proteini mogu odmah biti vidljivo i diferencira po elektroforeza metode. Theses gel tehnike takoðer se mogu koristiti za dobivanje malim kolièinama (micrograms) proèi¹æena polypeptides. Meðutim, oni ne pru¾aju velike kolièine proteina u njihovoj oèistili native dr¾ave. Substantial kolièine proteina oèistili , Od reda mnogih milligrams, potrebne su kako bi u potpunosti rasvijetliti njihove trodimenzionalni struktura i mehanizam njihovog djelovanja. Nekoliko tisuæa proteina su oèistili u aktivni oblik na temelju takve karakteristike kao velièine, topljivost, naplate i posebna obvezujuæa afiniteta. Na svaki korak u èi¹æenje, priprema je assayed za karakteristièan imovine proteina od interesa (npr. enzimska aktivnost) kako bi se ocijenilo je uèinkovitost postupka.

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. Proteini mogu biti odvojeni od malih molekula dijalizu kroz polu-permeable membrane, kao ¹to su celuloza membrana s pore. Molekula s dimenzijama znatno veæi od pore promjera su zadr¾ale unutar dijalizu bag, wwhereas manjih molekula i iona poprijeko pore takvog membrane i javljaju se u dijalizu izvan torbu.

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. Vi¹e razdvajanja diskriminirati na temelju velièine mo¾e se postiæi u tehnici gel-filtracija kromatografija. Uzorak je primijenjen na vrhu stupca sastavljena od porozne perle od jedan nerje¹iv, ali vrlo hydrated polimera kao ¹to su dextran ili agarose (koji su ugljikohidrati) ili polyacrylamide. Sephadex, Sepharose, i Bio-gel su uobièajeno komercijalnih preparata te kuglice, koje su obièno 100 mikrometar u promjeru. male molekule mogu unijeti ove velike perle, ali one ne mogu. Rezultat je mala molekula koja se distribuiraju i u voden otopina unutar kuglice i izmeðu njih, dok je velika molekula nalaze se samo u rje¹enje izmeðu perle. Large molekula toka vi¹e brzo kroz ovaj stupac i javljaju se prvi, jer je manji volumen je dostupan na njih. To bi trebalo biti navedeno da redoslijed nastajanja molekula iz kolone porous perle je preokrenuti u red u gel-elektroforeza, u kojem kontinuirano polimer okviru impedes pokreta velikih molekula. mnogo veæe kolièine proteina mo¾e biti odvojen od kromatografija gel filtracija gel by elektroforeza, ali po cijeni od ni¾e rezolucije.

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. The topljivost veæine proteina je smanjili na visoke koncentracije soli. Tom smislu, zove salting out, je vrlo korisno, ali nije dobro shvaæen. Ovisnost na topljivost soli koncentracija razlikuje od jednog do drugog proteina. Dakle salting out mo¾e se koristiti za fractionate proteini. Salting out je takoðer koristan za koncentraciji razrijediti rje¹enja proteina.

Copyright 2006 Molecular Station Copyright 2006 Molekularna kolodvor