How does the salting in/out technique separate proteins?
-> Salting in: As salt is added, solubility increases at low [ion]
-> Salting Out: in (aq) conditions at high [ion] , reduce solubility
= causes certain proteins to ppt.
How can we identify the correct technique to separate a specific protein?
Combining two protein properties makes it possible to derive a purification process
Outline the process of column chromatography
- Sample mixture dissolved in solvent poured in top
- Component with greatest affinity to stationary phase
takes longest to flow through - Each component collected as effluent reaches bottom
of column
(fractionated molecules)
What is chromatography?
Laboratory technique for separating mixtures in which components partition between a moving phase and a stationary phase
Why is ion exchnage often carried out as the first separation technique?
IEX separates larger amounts of materials than gel filtration
How are proteins eluted off the cation exchanger to ensure fractions are separated?
Column is washed with buffer to elute low affinity proteins
Why do we ensure the first wash in affinity chromatography isn’t too extreme?
Can cause the isolate to dissociate from the ligand, contaminating effluent
What are the disadvantages of HPLC?
- costly
- complex
- coelution
What are cation exchangers?
Acidic groups of resin that interact with positively (basic) charged proteins
How can elution be determined?
By [salt] of buffer or pH (stepwise/gradient elution)
In a chromatogram what does the area under the curve represent?
Area under curve = measure of [compound]
What stationary phase is used in IEX?
Stationary phase = polymer (matrix/resin) attached with charged groups
What is an elution volume (Vₑ)?
The volume of solvent required to elute a given solute from the column
What is the charge on the polymer beads in a cation exchanger?
Polymer beads have negatively charged functional groups
- (beads can be positively charged)
Which part of the purification process is GFC used?
GFC generally used near the end of the purification process
e.g. to separate correctly folded native protein from denatured protein
What biological molecules is affinity chromatography used to separate?
Antibodies
Antigens
Hormones
Other proteins
Why is chromatography significant?
It is important to isolate proteins in order to study their individual properties
What are the advantages of HPLC?
- speed
- high resolution
- sensitivity
- reproducible
- accuracy
- automation
How does GFC work?
Proteins separated based on size and shape
using a porous matrix to which molecules have different degrees of access
Larger molecules excluded form matrix first (elute faster)
Smaller molecules able to enter pores - take longer to elute
Which separation techniques are used to isolate proteins?
A number of separation methods are used in succession
- methods capable of using large quantities used first
- smaller quantity methods used last
e. g. mg
How is stepwise elution carried out?
- Protein mixture bound to upper most portion of ion
exchanger in coulmn - As elution progresses, various proteins separate into
discrete bands due to their different affinities for the
exchanger under the conditions - [salt] in the elution is increased to increase the mobility
- elute remaining bands
Why is it hard to observe the bands formed during HPLC?
Most compounds have no colour so can’t be seen by our eyes
The injected sample appears black to start off with and separates into yellow, red and
blue bands
How does ion exchange chromatography (IEX) separate proteins?
Proteins bind to an ion exchanger with different affinities
- the greater the binding affinity of a protein for the ion
exchanger column, the longer it takes to elute off the
column
How does HPLC enable a much better sample separation?
- solvent forced under 400 atm pressure through column
2. Allows use of smaller particles for column material (increases SA)
How does normal phase chromatography work?
Polar, solid stationary phase (e.g. Sillica)
Less polar mobile phase
- the more polar the analyte, the greater the retention time
How are molecules separated in affinity chromatography?
Molecules separated by taking advantage of their binding affinities for their respective ligands
What does a gel filtartion column look like?
Column consists of porous beads made of dextran or agarose
Outline the stages of HPLC
- Reservoir hold solvent (mobile phase)
- High pressure pump generates and monitors the
specified flow rate of the mobile phase (mm/min) - Injector injects sample into the continuously flowing
mobile phase stream - The mobile phase stream carries the sample into the
HPLC column - Detector detects the separated compound bands as
they elute from the column
How do we ensure all the unbound impurities are washed off in the first wash?
The first wash should have a sufficient [salt], pH or temperature in order to elute all
the unbound impurities from the solution
- need to be careful it’s not too extreme
What are the various components of HPLC?
- reservoir
- pump
- mixer
- injector
- column
- detector
- fraction collector
What are the different separating techniques used to differentiate between proteins?
- Salting in/out
- Chromatography (Column, Ion Exchange, Elution, Gel
Filtration, Affinity, HPLC) - Electrophoresis (IEF)
- Isoelectric Focusing (electrophoresis)
How is the elution of the hydrophobic bound proteins achieved?
By increasing [organic solvent], increases the retention time
How does the 2nd AC wash differ from the first?
Second wash needs to elute the isolate so must be significantly extreme in [ ], pH or temperature
Give an example of a cation exchanger
CM (carboxymethyl) cellulose formed via a reaction with alkali and chloroacetic acid
How do proteins interact with the reverse phase stationary phase?
All proteins contain hydrophobic and hydrophilic amino acids
- those with high net hydrophobicity participate in
hydrophobic interactions with the stationary phase
How does chromatography work?
Samples are dissolved in a mobile phase (gas/liquid) then forced through a stationary phase
How are proteins separated using 2DE and SDS?
- Isoelectric focusing occurs
- Isoelectric focusing gel placed on SDS polyacrylamide
gel - SDS PAGE electrophoresis - protein subjected to
electric current perpendicular to IEF
Why are proteins able to be separated using affinity chromatography?
Proteins can bind specific molecules very tightly but not covalently
What is HPLC?
High performance liquid chromatography
How does each technique differentiate between samples?
IEX - based on charge distribution GFC - separates on molecular size AC - binding properties IEF - based on pI HPLC - hydrophobicity and high pressure
How does Reverse phase chromatography separate proteins?
Separates molecules based on their hydrophobicity
using a non polar stationary phase
and a polar mobile phase
Polar proteins elute first
Non polar proteins bind to column - longer retention time
Outline the stages of affinity chromatography
- Protein mixture added to column containing polymer
bound ligand specific for protein of interest - Unwanted proteins are washed through column
- Ligand solution poured in
- Proteins of interest eluted by ligand solution
How is gradient elution used to increase the quality of separation?
The mobile phase is progressively changed
This increases the [organic solvent]
Causing a decreased retention time
- hydrophobic proteins bind to hydrophobic stationary
phase
- hydrophillic proteins don’t bind and wash out
How are proteins separated using a cation exchanger?
- Protein mixture added to column containing cation
exchangers - Proteins move through column
- rate depends on net protein charges at the pH being
used
- rate depends on net protein charges at the pH being
- Proteins with -ve charges move faster and elute earlier
with cation exchangers
What is the reverse phase mobile and stationary phases composed of?
Stationary Phase
packed with silica modified with silyl esthers containing non polar
alkyl groups (c18 or c8) - creating a hydrophobic end
Mobile phase
contains organic solvents (e.g. methanol or acetonitrile)
What is a common use for gel filtration chromatography (GFC)?
Separating complex samples of biological molecules e.g. blood
Can use GFC to determine molecular weight of molecules
How do we remove the protein of interest from the column?
Use a higher affinity for ligand (competitive) solution
Can alter pH, ionic strength and/or temperature to destabilise the protein-ligand complex
What are anion exchangers?
Basic groups of resin that interact with negatively charged (acidic) proteins
How does IEF + SDS-PAGE electrophoresis separate proteins?
2D gel electrophoresis (IEF) separation is based on osmotic potential
Followed by SDS-PAGE which is based on protein size
What basis are samples separated on in GFC?
Based solely on molecular size
What components are required for affinity chromatography?
- beads matrix (agarose in column)
- solution containing substance to be isolated
- ligand (binds specifically to protein)
- wash to elute non bound substances
- final wash containing competitive ligand
What factors affect a proteins affinity for the stationary phase?
Proteins contain multiple charged groups which alter affinity
Solubility effects affinity
What factor does Reverse Phase Chromatography use to separate molecules?
Reverse Phase Chromatography is separation based on protein solubility
Why is sillica a popular choice of material for the stationary phase?
Readily available
Low cost
OH- group on surface increases polarity
What is Reverse Phase chromatography?
The most widely used mode of high performace liquid chromatography (HPLC)
What does a proteins solubility depend on?
Solubility depends on:
- [dissolved salts]
- solvent polarity
- pH
- temperature
How is the affinity material prepared?
Inert support + spacer arms + ligand –> affinity material
How can proteins be selectively precipitated using salting in/out?
Can alter the type of salt and [salt] to precipitate certain molecules
Name an anion exchanger used in IEX
Diethylaminoethyl (DEAE) cellulose