Protein Gel Electrophoresis Flashcards
What are some of proteins crucial roles in biological processes ?
- Transport and Storage
- Mechanical support
- DNA/RNA metabolism
- Enzyme catalysis
- Control of growth and differentiation
- Immune response
What do proteins differ in ?
- Size
- Shape
- Charge
What is Electrophoresis ?
This is the process by which large charged molecules travel through a matrix under the influence of a uniform electric field
What does the matrix act as ?
It acts as molecular sieve to aid separation
What are some examples of these matrix ?
Polyacrylamide gel, agarose gel, paper and cellulose
Polyacrylamide gel electrophoresis (PAGE)
How is polyacrylamide gel formed ?
By polymerisation of acrylamide
Explain PAGE ?
- Mesh-like fiber network in three dimension
- Creates an environment that allows different sized proteins move through the gel at different rates
How does PAGE separate proteins ?
According to their molecular weight
However, proteins have different ?
They are different shapes, and have different charges
What does SDS-PAGE stand for ?
Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis
What is the aim in an experiment involving SDS-PAGE ?
To separate proteins according to their size i.e. chain length
What is the problem ?
Proteins have secondary, tertiary or quaternary structure → wide range of shapes and charges
So what is the solution ?
Use SDS to convert all proteins to the same shape and mask net charges
What does an Anionic detergent do ?
- Disrupts non-covalent interactions (hydrogen bonds, hydrophobic interactions) thus unfolding the protein
- Binds most proteins in a constant fashion (about 1.4 grams of SDS per gram of protein)
So, what does this ultimately do ?
- Masks net charge of the protein by forming large anionic complexes
- Minimises differences based on secondary or tertiary structure
Why are reducing agents added to do ?
- Break disulphide bonds
- Detach subunits to ensure a separation based only on molecular weight
So what does this ultimately to do the proteins ?
- Proteins have an homogenous negative charge
- Move to cathode when electric field is applied
- Larger proteins are more retarded.
Gel has two layers that differ in their acrylamide concentration and pH. Explain them ?
- Stacking gel:
low concentration acrylamide (4%), low pH (6.8) - Resolving gel:
higher concentration of gel (10-20%) and high pH (8.8, same as tank)
What needs to be done so all proteins all enter the separation gel at the same time ? resulting in ?
Concentrate proteins in a narrow band in the interface between gels
This will result in a greater resolution of bands
One acrylamide concentration only separates a fixed range of protein sizes: hence ?
Use a gradient for a broader range of separation
The proteins migrate through ?
An increasing percentage of acrylamide (e.g. 4-20%)
Thus the gel becomes increasingly ?
Restrictive for the proteins to migrate
Most commonly used in ?
Lab, commercial pre-cast gels
Small proteins run too fast in ?
Low percentage gels
Gradient gels can separate?
A broader range of protein sizes
Once the gel is run we need to visualise the proteins:
- Fix (immobilise) the proteins with a weak acid: proteins become insoluble and are thus prevented from diffusing during staining
- Stain with Coomassie blue (or similar) dye to make protein bands visible
- Then destain to remove excess dye
Why use Non-denaturing gel electrophoresis (native-PAGE) ?
Separation according to:
- Size
- Net charge
- Shape of native structure
Native vs Denaturing gel run ?
In Native gel - All four levels analysed as secondary, tertiary and quaternary structure maintained
However, denaturing gel (e.g. reducing SDS-PAGE) - Primary structure analysis only happens
What is Western blotting used for ?
Technique for identifying a particular protein using antibodies after electrophoretic separation in a gel and transfer to a membrane.
Explain the three steps ?
- Separate proteins using SDS-PAGE
- Transfer into nitrocellulose membrane
- Stain and visualisation using antibody against protein of interest
Why and how is two-dimensional gel electrophoresis used ?
- Thousands of proteins in cells: too many to separate by SDS-PAGE gel alone
- Use two dimensions, to separate the proteins on the basis of:
- isoelectric point in the first dimension: isoelectric focusing
- molecular weight in SDS-PAGE in the second dimension
Low pH ?
Acidic groups neutralised
High pH ?
Basic groups neutralised
Change the pH, and what changes ?
The net charge changes
What is the isoelectric point (pl) ?
pH at which the protein is neutral
When is this different and what does this depend on?
- Different for different proteins
- Depends on amino acid composition
At all pH values other than their pI, proteins will be?
Charged
- at low pH, the protein is positively charged
- at high pH, the protein is negatively charged
Proteins accumulate at a pH region that corresponds to ?
Their pI, where they have no net charge and migration ceases.
Result: proteins focused into ?
Sharp stationary bands with each protein positioned at its pI.
At the isoelectric point the protein has no ?
No net charge and therefore no longer migrates in the electric field; for the protein shown the isoelectric is 6.5
Explain Protein identification ?
- (Western blotting or)
- Cut protein band out of gel
- Digest with protease to make peptides
- Identify by mass spectrometry
What is Relative mobility (Rf ) ?
This is the distance migrated by a band divided by the distance migrated by the dye front (i.e. a small negatively-charged molecule).
How can we measure Relative Mobility of Protein Bands ?
- Related to the protein’s molecular mass
- Run a set of protein standards of known molecular mass
- Draw a standard curve of log (mass) versus relative mobility
- Estimate apparent mass for unknown proteins
