Chapter 6 isoelectric focusing Flashcards
Objectives
Understand the principles of isoelectric focusing (IEF)
Understand how a pH gradient is formed
Understand advantages of immobilised pH gradients (Immobiline)
Understand the techniques used in IEF experiments and applications of IEF
Principles of IEF
An electrophoretic process, in which proteins are separated according to their isoelectric points
Regardless of the point of loading, proteins are “focused” to seek their isoelectric points Isoelectric point (pI) is the pH at which a protein has a net charge of zero.
If the pH is less than pi
the protein becomes positively charged, which makes it attracted to the negative end of the strip, low pH
If the pH is more than pi
the protein becomes negatively charged, which makes it attracted to the positive end of the strip, high pH
How being amphoteric affects the protein
Depending on the pH of the environment, each protein can be overall positively or negatively charged or have zero net charge.
Relationship between charge and pH
Overall charge of a protein is dependent on the pH of the surrounding environment
pH > pi, proteins are negatively charged
pH < pi, proteins are positively charged
pH = pI, proteins have no charge
Why form pH gradient
a pH gradient needs to be established in order for proteins to seek out their isoelectric points
What are the 2 types of reagents used to generate a pH gradient?
- carrier ampholytes
2. immobiline reagents
Carrier ampholytes use
Used in conventional IEF
Immoboline reagents use
Used in immobilined pH gradient IEF
Carrier ampholytes principle
They are low molecular weight (400-1000Da) zwitterions at a certain pH and buffer at that pH
Synthetically-made molecules to comprise a range of pI values.
When an electric field is applied, carrier ampholytes will arrange themselves to build up a pH gradient.
Comes in a range of pI ranges to achieve optimal resolution.
Role of carrier ampholytes in IEF
form a pH gradient when added
Ampholytes and gel matrix
4 steps
Ampholytes are not well integrated with the gel matrix
- an ampholyte solution is incorporated into a gel
- a stable pH gradient is established in the gel after application of an electric field
- protein solution is added and electric field is reapplied
- after staining, proteins are shown to be distributed along pH gradient according to their pi values
2 disadvantages of conventional IEF system
- carrier ampholytes are more mobile than proteins
, thus IEF needs to be continued after pH gradient is set up - need for optimization of focusing time after pH gradient is set up, otherwise the pH gradient collapse due to cathodic drift
cathodic drift problem
cathodic drift, where the pH gradient decrease over time, may occur if a gel is focused too long.
Cathodic drift is observed as focused protein migrating off the cathode end of the gel.
Electro-osmotic flow
Movement of water (H3O+) to cathodic end, carries with it basic ampholytes and proteins
A result of prolonged IEF
More than standard duration of usually 3hours
Main cause of cathodic drift
Electro-osmotic flow
How to prevent pH gradient collapse
What if the pH gradient can be pre-formed and immobilized on a gel?
How to immobilize pH gradient
Immobiline (IPG)
Immobiline (IPG), use of acrylamide monomer
Use of weak acidic and basic buffering reagents, covalently bound to an acrylamide backbone
Mixing different proportions of the weakly acidic and basic buffering reagents result in “carrier ampholytes” buffering at different pH.
Titration of the weakly acidic and basic buffering reagents set up a pH gradient during gel casting.
The backbone of the reagents is an acrylamide monomer, allowing polymerisation to take place with the gel.
Polymerization immobilizes pH gradient
Immobiline dry strip
commercially available
can choose pH range
can choose length of the strip
immobline reagent
acidic and basic buffering reagent that we use to immobilize the pH gradient
what happens when immobiline reagents are integrated with IEF gel
Incorporation of different amounts of amine and carboxylic groups covalently bound in result in different localised pH environments, thus create pH gradient
2D-PAGE using IPG DryStrip
the stacking gel region is not the usual stacking region, it is tier spaced, allow us to transfer the strip once we completed the first dimension
carry out the normal SDS-PAGE, proteins will now be resolved based on the molecular weight
The stacking gel in 2D-PAGE using IPG DryStrip
Stacking gel (%T=4-6)
IPG DryStrip (T=4%,C=3%) replaces the stacking gel, (enable loading of proteins at high concentrations)
The resolving gel in 2D-PAGE using IPG Dry Strip
Resolving gel (%T fixed between 10-15)
Separates proteins by MW
There are different types of dry strips
vary in pH ranges and length, depend on sample we work with
Choice of pH range
depends on sample, analysing protein from cell lystae, using broad pH, better separation of all proteins
separating proteins characterized by pH and Pi, narrow will more suitable
non-linear drystrip basically allow more even distribution of proteins of interest
broad pH range uses
Broad pH range gel strips allow separation of most protein mixtures from prokaryotic and eukaryotic sources.
narrow pH range uses
Narrow pH range gel strips allow better resolution of proteins with a known pI range, especially those that do not resolve well on a broad range gel.
Non-linear pH range uses
Gel strips with nonlinear (NL) pH ranges allow a more even distribution of proteins along a specific pH range to maximise resolution.
Round 1
Broad pH range for maximal resolution of all proteins
Round 2
Narrow pH range to better resolve particular group of proteins
2D PAGE
the blue streaks and spots correspond to stained proteins
use a narrow pH range subsequently after broad pH range to separate the proteins better
Linear gel
Linear pH gradient over the whole pH range
Non-linear gel
Increased resolution between pH5 to 7 with a non-linear pH gradient
It can separate proteins better. Proteins are much more well separated than when we use non-linear gel
Estimating pi of individual protein spots
we can also use gel strip to determine pi of the respective proteins spots
measure length of strip
determine the distance of protein spot of interest
express the 2 above as a percentage
The pI of the protein spot can be estimated by relating the position of the protein in the 2D-PAGE gel to its original position in the Immobilline drystrip.
Plot a graph displaying pH gradient of the strip vs the percentage of the gel
Read of the graph
y axis is pi range
% gel length is x axis
read off the 2
immobiline in dry spots
Besides non-linear gel strip to improve the resolution of protein spots, use gel strip with overlapping ranges
Improving resolution using “composite maps”
Protein separation is not too ideal when it moves to the right, a bit cluttered. If we are interested in those proteins, run a gel from 4.5-5.5, allow region from 4.5 to be better separated as compared to the original gel from 4 to 5
advantage of using overlapping pH ranges, help to improve the resolution
2 steps principle of Improving resolution using “composite maps”
Creation of ‘composite maps’ with overlapping pH ranges.
Enables better resolution of low abundance proteins.
How to determine length of drystrip to use
as we improve the length of the strip, the time to carry out the 1st dimension also increases
Advantages as the length of strip increases
Sample loading capacity increases
Resolution of proteins increases
Number of spots detected increases
Disadvantage as the length of strip increases
Focusing time increases, more time needed for focusing
Cost effectiveness decreases
