protien purification

what does protien purification require

seperation of the protien you wan t from others

what does SDS - PAGE stand for

sodium dodecyl sulfide (a detergant) and polyacrylamine gel electrofresis

what is SDS PAGE

an analitic technique

whats the role of SDS

unfold the protien with the aid of boilin this makes it liniar and coated

what the result of the SDS

it means tha any inherent charge is stamped

what is the PAGE component

the protiwn is run through a gel electrofresis - smaller molecules move futher

what can the PAGE be used for

incluson of marker protiens with a known molecular mass enables us to identify the molecular mass of the protien

how is the molecular mass of the protien found

using a log graph og molecular mass vs mobility

why is this olny used as a purification thechnique

caun use the proties as there were denatured when boiled

what diffrent structures can protiens be speperated by

solubility, isoelectric focusing and columb chromatography

how is solubiltiy used to seperate protiens

protiens are salted out using ammonium sulfate, when the protiens have decreaced water avaliabitity they will form a preticipate when centrafuged

what does isoelectric focusing use

imobilised ph and electric feild

what is the isoelectri point and how does it seperate the protiens

when the molecule has no net charge, they willl move alon the strip untill their isoelectric point and then stop moving

what can isoelectric focusing be combined with

SDS PAGE the strip can be placed in the gel andf the protiens run through

what are the diffrent methods of columb chromatography

size exclusion, ion echange and affinity

explain size linked chromatography

there are lots of cross linked molecules with diffrent sized pockets, the smaller molecules fall into the pockets and so take longer to get to the bottom than the larger ones

what can size exclusion chromatography be used with

a callibration curve to work out native mass

what does ion exhange chromatography rely on

the charged groups on protiens, ph is important

describe ion exchange chromatography

polymers are substituted with charged groups - anion and cation. charged groups interact with matrixes and get stuck

how can the protiens be knoeck off of the matrixes, what is this dependant on

run through Na Cl to knock off protiens dependant on weakness

what is affinity chromotography dependant on

most protiens have selective affinity to particular structures

describe the prosses of affinitive chromatography

the interacting unit is put in a columb - the protien is run down it an will stick

how is the protien then removes form the interacting molecule

a pH diffrence or a ligand added free that will compete and cause the protiens to become free

why is affinity chromatography useful

whe can desighn it via gene manipulation for any protien we want