chromatography Flashcards
solubility
proteins are soluble only in a limited range of salt concentration and pH
solubility depends on the concentration and type of salt. the pH of the solution(solubility always lowest at the proteins pI = 0 net charge (high conc. of some salts stabilise but rest cause denaturation), the number of hydrophilic residues on the proteins (the more the better -= hydration layer), and the number of hydrophobic residues (increases as salt conc increases = reduces solubility + hydrophobic patches stick together + precipitates out of solution)
solubility always lowest at the proteins pI = 0 net charge
different proteins in same salt behave different
same protein solubility in diff salts = diff solubilities due to salt specific properties
type of salt (anion and cation)
ion size and water binding properties influence solubility
ammonium sulfate salt precipitation
soluble proteins can be fractionated by salt precipitation but salt needs to be removed after
ammonium sulphate stabilise protein structure to make them insoluble and allow selective precipitation
as u increase [(NH402SO4)] solubility decreases with increasing ionic strength
using 40-60% salt
1. adjust ammonium salt conc so that the POI only just remains soluble but some protein contaminants will precipitate
- centrifuge - protein stays in solution - pellet of contaminants removable
- add more salt to supernatant to just precipitate the POI - ammonium sulphate will bind to the proteins, clump together and precipitate out
- pellet protein and retain it and discard the supernatant containing other contaminants
up to 5x purity increase - dialysis tubing is selectively permeable to low molecular weight i.e contaminants
i.e. ammonium sulphate salt + protein retained inside tubing
when put in buffer filters protein until equilibrium reached
salting in
salts at low conc - makes them more soluble
increasing the solubility of the protein at low ionic strengths
at low conc of salt, shielding the proteins from each other= inter molecular shielding of charge charge interactions
the salt will bind the anions will pined to positively charge amino acids on protein = shield the proteins from interacting with each other and becoming insoluble = ionic attraction between protein molecules reduced = increases solubility
salting out
at high salt
the salt will replace the outer hydration layer os the protein and exposes hydrophobic patches on the protein
decreasing solubility - precipitating the proteins at high ionic strength
lose water molecules the are sheilding the protein
hydrophobic patches come out
hydrophobic interactions between proteins are increased = protein will clump together decreasing their surface area - protein falls out of solution
gel filtration chromatography - separating by size
size exclusion chromotography
porous polymer beads (stationary phase - as liquid inside the bead is immobile) allows proteins to enter the beads only if they’re smaller than the pores - larger are excluded and travel around the beads and emerge first = at the Void volume = Vo
medium diffuse into bead so are slower than the larger proteins
small proteins visit all the available volume inside the beads so elute the slowest at the column bed volume Vt
HPLC - reservoir of water (buffer) has a pump to introduce it into the column
mixture of proteins injected into column using a syringe
large molecules pass through earlier into the fractions
the eluent is outside the bead and flows around not through beads
Void volume - liquid outside beads Vo + (internal bead volume Vi = stationary beads) = bed volume Vt
Vt =Vo + Vi
must calibrate the column to measure where our unknown protein comes out
partition co efficient = Kav is the fraction of molecules that can enter a bead compared to the fraction total
if protein never enters the stationary phase too large gets excluded Kav = 0
and it elutes it at void volume
protein freely penetrates the bead
[stationary phase] = [mobile phase] - so Kav = 1 and the protein elutes at the total column volume (Vt)
other proteins partite penetrate and have Kav between 0 and 1 so elute somewhere between Vo and Vt
proteins emerge according to their partition coefficient Kav
- have a specific Ve value
to obtain apparent molecular eights must calibrate coolumn using proteins of known molecular weight. then rerun the same column loading only the unknown protein. measure its elution volume and use the calibration graph to obtain its molecular weight
assuming the unknown protein is globular like the known proteins and that theres not chemical interactions between the beads and proteins and simple just a partition co efficient which would case the proteins to be retained longer as give a lower apparent molecular weight. proteins may form complexes homo and hero dimers eluting earlier and giving higher apparent molecular weights. can check by SDS-PAGE as protein is denatured and can measure its true molecular weight
ion exchange
separation by the net charge of a protein
HPLC system like in gel filtration
positively or negative charged chromatography media resin - anion or cation exchange chromatography
those that are opposingly charged and are large elute first
reversible displacement of ions that are bound to a charged matrix in adsorption
anion exchange =
net positively charged bead attracts negatively charged anions = exchangeable counter ions
Weak AEX resin - DEAE diethylaminoethyl
Weak CEX resin - carboy methyl CM
how to get the proteins off after the protein selectively binds
overwhelm w high concentration with further ions
NaCl to elute proteins from strong IEX resins
buffer pH and pI of protein of choice determines Resin AEX or CEX choice
net protein charge depends on buffer pH because as you move the pH you can ionise certain groups or render them. neutral
if the buffer pH id below the pI (higher [H+]) the protein is positive (a cation)
and binds to a negative CEX resin CM)
so if pH of the buffer is above the pI, (lower{h+] the protein is negative (an anion) and binds to a positive AEX resin (DEAE)
if buffer pH is equal to the pI the protein has no net charge and does not bind to any ion exchange resin
in an AEX
when mixed sample of molecules is poured in the column the slightly negatively charged ions will bind preferentially to the positively charged beads but any ions that are not very strongly negatively charged will bind very well. as more solution is pumped in, the initial cloud of ions moves down and off of the column and negative proteins adsorbing their place. as the negatively charged ions increase in concentration chloride ions get pumped into the column they start to compete for the proteins that are on the column, the weaker negatively charged proteins will desorp off first as the stronger ones will hold on more tightly so for longer
AEX resin
loading:
at high pH
negatively charged proteins anions displace exchange bound negatively counter ions and bind to the resin
positively or zero charged proteins pass straight through and are collected = non binding fraction
elution
proteins are displaced and exchanged with negative ions by increasing salt concentration as a gradient
counter ions compete with the protein for the charge on the resin
higher salt conc are needed to release and elute the most negatively charged proteins
reverse-phase chromatography
bind hydrophobic molecules very strongly opposing normal chromatography media which bind hydrophilic proteins
if proteins had hydrophobic patches on it i.e. isoleucine, valises tryptophan, binds
stationary phase - solid silica beads with non polar hydrophobic hydrocarbon chains of different length covalently attached
mobile phase = water-acetonitrile (AcN) organic non polar solvent but soluble in water small hydrophobic molecule
in excess conc able to elute hydrophobic proteins back off of stationary hydrophobic column
problem - can denature protein so often used for DNA instead
mixture is poured and partition onto the chains
very hydrophobic molecules will bind to C18 and not so hydrophobic will bind less tightly
separates by hydrophobicity and is entropy driven process as water is initially in clouds around hydrophobic patches and molecules as they bind drive off that shell of water releasing it into the solution driving the binding of protein of interest
the longer the side chain length the higher the [AcN] require for elution as hydrophobicity increases
C18 forest of alkanes bound to silica
proteins have ordered water and have hydrophobic patches that are exposed that want to get buried in hydrophobic column
water is driven off helping protein bind to column = adsorption phase of proteins to hydrophobic column
to release protein by increasing [AcN]
many proteins have few exposed hydrophobic groups so bind poorly to HPLC resins
to improve binding…
mixture of charged proteins separating out by neutralising some of their charged groups using TFA an ion pairing agent
forms stable salt bridges = neutralising leucine and increasing the hydropho
proteins elute in order of increasing hydrophobicity
higher {AcN] required to elute proteins from a C18 column than C8
affinity chromatography - bio-specificity
two halves
buffer with a pump that res the buffer in and mixture injected into the column
immobilised ligands covalently attached to beads
active site/ binding part of protein can selectively latch onto yourligand and effectively immobilise temporarily your proteinon the surface of the bead while all other molecules which can’t bind ligand flow through and out the column
elute using high conc of free ligand into top of column to compete with the immobilised ligand as conc of free ligand will overwhelm POI causing it to move off
binding non -covalently to other smaller molecles
strong reversible ligand interactions are used to selectively purify only that protein
selectivity and speed
identify biological affinity that your protein has with a ligand
protein ligand interaction if it has a low Kz = specific tight interaction
immobilise ligand covalently onto bead support column
pour sample
and then desorb proteins by increasing sodium chloride conc/free ligands to break protein-ligand interaction
fusion tags aid purification as they are recognised by the immobilised ligand - proteins that are difficult to purify can be produced as a recombinant fusion covalently linked to a tag
i.e. histamine tag which bind to a nickel column useful too
the tag is added at the C terminal
then express fusion protein in E coli cell using IPTG induction
then lyse the cells = cell extract
bound protein is led from the column with an imidazole gradient
SDS page reveals which fractions contain the target protein
the non binding fraction contains none of the POI
you need to do dialysis after each type of chromatography
chromatography exploits the differing biophysical properties of protein to separate them
some combination of techniques is required for a pure sample
