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?