Steven Matthews - Protein Purification Flashcards
General Objectives of Lecture Series? How does it link in with previous content?
From a historical context, how has protein purification evolved?
What are the main factors that are exploited when performing protein purification?
Why do we want to purify proteins?
What the salting out - purification technique?
What salts are common used for salting out? How do we know whether one salt/ion works better than another?
How does adding salt to the solution encourage partitioning in salting out?
What is protein chromatography? What are the two general categorizes?
Protein chromatography - Seperating proteins based on the fact thath they move at different rates through a column
- Absorption/Desorption - Requires a solid phase (column) that will interact with a soluble protein (liquid phase).
Both solid-liquid phases are dependent on the thermodynamics of the interaction of soluble proteins with a solid phase - important to understand the thermodynamics of this interaction to optimize purification
- Permeating - Pure liquid-liquid phase - method is dependent on the rate of diffusion (kinetics) between liquid phases
What are some experimental setup and considerations that one needs to consider for both Absorption/Desorption and permeating chromatography?
What does adsorption and desorption to a column refer to? What factor dictates whether one or the other happens?
Outline the Langmuir Isotherm equation and all it’s variables? Why might we want to change it?
What easily predicted variables can we substitute in into the Langmuir Isotherm to make it more user friendly?
How can we manipulate to Langmuir Isotherm to show the fraction of protein bound rather than fraction of binding sites occupied? What do we end up with?
Breakdown of the modified Langmuir Isotherm?
The equation now has two variables which we can exploit..
- Γ is the total concentration of binding sites which we can decide when we order the column
- Kd is dependent on the type of interaction –> which is dependent on solid phase and protein interaction - can be manipulated
Remember α is the key quantity as it represents the fraction of protein bound by the solid phase –> what we want o find out…
The equation implies good adsorption (protein binding) depends on…
- High concentration of binding sites (Г) - higher probability of binding
- Low dissociation constant (Kd) - more remains on column –> tight binding
Solution conditions can be changed (pH, I, polarity) –> influencing Kd and hence α
For general chromatographic separations by adsorption what values of alpha do we use when we apply and elute our protein from the column?
In our modified Langmuir isotherm, what variable influences α? Why might it be misleading/inaccurate?
On a chromatographic column, is Adsorption/desorption instanteneous?
No, Adsorption/desorption is not an instantaneous process
The protein needs a certain amount of time in contact with the solid phase to reach equilibrium
Reach a balance between flow rate and equilibrium
We want to adequete flow rate to allow our proteins to seperate but at the same time we want to allow our protein to interact with the column to allow for adequete seperation
On a chromatographic column, what does dispersion refer to?
How does diffusion contribute to band spreading?
How to find out dispersion and resolution from a Gaussian shaped elution profile?
What is one important thing to remember about band spreading?
We intially stick the protein to the column - high alpha value.
Only once we decrease the the alpha value - begin elution - will we observe the band spreading
Hence, our protein of interest will not be the only protein in the column once elution occurs - we want to do as much as possible to improve our resolution!
What is turbulent flow?
Apart from diffusion, turbulent flow is the other factor that contributes to band spreading.
Breakdown the following equation - what can we learn from it?
This equation helps us decipher all the optimal conditions to minimze dispersion (sigma) - empirical equation (based on experimental data)
What the self-sharpening phenonmenon that is observed as proteins move through the column.
The theory suggests that as the band moves through the column it should self-sharpen - concentrate itself
This means that…
The Middle of the band is accelerating and catching up with the front of the band –> results in self-sharpening
Is this all good?
Not completely, since other protein bands may catch up with the tailing end of the band in front
Why does self-sharpening occur?
In order to understand why self-sharpening occurs we need to look at the impact of total protein concentration on the distribution and binding.
Note - We did remove Pt (total concentration) previously but even though binding sites are in excess – PT has a small but noticeable impact –> leading edge sharpens up (higher conc.) and trailing edge spreads out.
Here is why?
- Higher concentration of protein –> slightly weaker binding –> meaning that the middle of the band (highest conc) moves faster and can catch up with the front
- Lower concentration –> stronger binding –> meaning at the back of the band, where we find a low concentration, binds to the column with a higher affinity, thus moving slower –> resulting in tailing?
Mathematically –> Shown in image
Conceptually –> fewer protein molecules exposed to binding sites - less competition for binding - statistically more likely to bind
What are the four types of adsorption and desorption chromatography?
What is ion-exchange chromatography? What are some requirements for the solid phase?
What are some typical substituents that are derivatized to the column?
Outline the general procedure of applying your protein to an ion-exchange column? What two variables can be manipulated to acheive elution?
Normally exploit Ionic strength and use a single buffer with a single pH
When running ion exchange chromatography, at what ratio will the protein titrate at, given that the charge does not change?
As there is usually an excess of binding sites over protein concentration, pH changes do not have much effect on capacity (column itself) but will affect α (fraction of protein bound)
Assuming charge on protein does not change (NOT true near pI)…
α will titrate with the ratio of charged (binding) to non-charged (non-binding) proteins
Charged groups bind to column (don’t elute) whereas, uncharged groups do NOT bind (will elute)
Hence, Alpha is dependent on the charge - charges of proteins change so will alpha
Takeway - Titration (slowing changing) of protein groups’ charge is usually more important than the binding sites (especially true for strong exchangers) and this will affect Kd
How can we calculate how many charges we need on a protein for it bind to a typical column?
Each favourable charged interaction between protein and column contributes roughly 1-2 kJ mol-1 to the binding energy
6 kJ mol-1 is required to change Kd (k) by a factor of 10 (1.5 kJ mol-1 average) –> Corresponds with a change in protein charge of 4 which is sufficient to alter overall binding - switch from binding to elution (alpha = 0.5)
This is just a simple way to examine how changes in pH effect Kd, which in turn impacts alpha and dictates how much protein is eluted.
When running ion-exchange chromatography, what other factors need to be taken into account?
What are the key things you should take away from the attached image of a modern chromatographic sampler?
What is the phenomenon of band sharpening in chromatography?
What is chromatofocusing?
How do we create the pH gradient needed for chromatofocusing?
When eluting using a pH gradient, what order will the proteins elute with?
In general proteins elute in order of, but not necessarily, their pIs
This is because pI is an equilibrium quantity (thermodynamics) whereas elution is a dynamic quantity (kinetics)
What is Hydrophobic Interaction Chromatography (HIC)? How does it work?
Hydrophobic patches can also be found on the surface –> But we need to be careful not to denature our protein as the hydrophobic core is extremely hydrophobic may want to bind with hydrophobic column.
Similar to ion exchange but based upon hydrophobic interactions –> Solid matrix carries bound aryl or alkyl groups:
Phenyl – moderate
Octyl – strong
General procedure
- Proteins are loaded in high salt to promote the ‘salting out effect’
High salt concentration promotes salting out - encourages hydrophobic regions to interact
- Proteins are eluted with a decreasing salt gradient - resulting in the reverse (salting in)
Highly hydrophobic protein can be eluted with an organic solvent/H2O mixture (ethylene glycol or methanol)
Problem?
Potential denaturation
What is Reverse Phase Chromatography?
What is the basic idea about affinity chromatography? What are some factors that lower its efficiency?
What are the two broad types of ligands used in affinity chromatography? Also give five different types ligand examples?
What is a common method of attaching the ligand to the column carbohydrate matrix?
Apart from CNBR attachment, what other methods of attachment are available?
How do you elute using affinity chromatography?
Outline the gel-filtration procedure.
What happens during gel filtration? How does seperation occur?
How to predict Mw of the species via the gel filtration column?
What purification technique is normally done first, second and last?
- Affinity - almost always the first step - most selective
- Ion exchange/hydrophobic interaction - in the middle
- Gel filtration last –> least selective
Low capacity column, no binding and we need to start with a concentrated protein solution
Longer column best - allows time for this to happen
What is a purification table?
How does protein electrophoresis fit into the purification procedure?
Previously we examined different techniques to purify as much native protein so that it can be analysed further (biochemical, structural, therapeutic, etc.)
Protein electrophoresis is used more as an analytical technique –> examine the success/purity of each purification technique
Visualize how pure and impure our sample is + also estimate measure protein concentration
You tend to not use protein from protein electrophoresis for further downstream analysis
What is protein electrophoresis? What is the rate of migration (M) proportional to?
High Ionic Strength - proportion of current carried by ions in buffers increases but results in decrease current carried by sample - decreasing rate of migration
What are the 4 principle types of protein electrophoresis?
What is the polymer used in protein electrophoresis?
What are the two main ways to detect/stain proteins in electrophoresis?
Detection:
- Coomassie blue dye (~1 𝛍g of protein per band)
- Silver (1 ng of protein per band)
How are proteins denatured for a denaturing gel?
Outline the setup used for a discontinuous gel - protein electrophoresis.
Tris/Glycine buffer
What is the charge of a glycine zwitterion at pH 6.7 & pH 8.9?
How does the stacking gel concentrate the protein’s in our solution?
Stacking gel - high porosity - no seperation by size (5%)
Running Gel - low porosity - seperation of protein by size
The glycine and Cl- form a sandwhich around our protein - arises due to differetial charge - remember the protein is negatively charged as it is surrounded by SDS
What happens when the protein bands enter the running gel? What is the data produced by the running gel used for?
What is native gel electrophoresis? WHat is it used for? Setup information?
What is isoelectric focusing?
What is 2-Dimensional (2D) Page?
Advantages and disadvanatages of Capillary Electrophoresis? What are the requirements?
What are the two elements driving movement in Capillary Electrophoresis?
Outline how electro-osmosis works in a capillary tube?
What does the following force diagram show?
What is Capillary Isoelectric focusing?
