Gel Electrophoresis Flashcards
What is gel electrophoresis ?
Defined as the separation of molecules based on migration in an electromagnetic field
Uses 2 electrodes - anode (+ve) and cathode (-ve) and
an electrolyte gel which serves as a conducting medium
The rate and direction of the particles moving within the gel will be dependent on their size and molecular charge
What can be separated ?
Anything which possesses ionisable groups which at a given pH in solution exist as an electrically charged species
E.g amino acids, nucleotides and nanoparticles
Describe the Agarose Gel Electrophoresis technique:
Molecules to be separated are pulled by an electrical field (negative to positive) through a gel containing
small pores - agarose
Molecules travel through agarose at a speed inversely related to their length
Smaller DNA molecule (less -ve charge) will travel further
Describe the gel age of agarose:
0.7% - 2.0% agarose
0.7% - good separation of large DNA fragments
2.0% - good separation of small DNA fragments
Describe the visualisation of agarose including advantages and disadvantages of new products:
Visualisation dyes can be added before, during or after the gel is run
Previously used Ethidium Bromide and UV light but now much safer molecules are used (less teratogenic and mutagenic)
Now use SYBER safe or GelRed®
Advantages –
relatively easy, cheap and fast to perform, can give you a rough size of product
Disadvantages – semi quantitative as you can’t determine the amount of product generated easily
Describe the Polyacrylamide Gel Electrophoresis (PAGE) technique :
Used to separate proteins or fragments of DNA <1 kb
Formed by the polymerisation of acrylamide + N,N -methylene-bis-acrylamide (the cross-linking agent)
Polymerization initiated by addition of ammonium persulfate + DMAP or TEMED
2 types;
- native non denaturing PAGE
- denaturing PAGE
What is native non-denaturing PAGE ?
Used to separate proteins/ nucleic acids based on their size, shape, and charge, without disrupting their natural structure or function
Mobility depends on protein charge & hydrodynamic size
Possible to recover proteins in their native state
Protein-protein interactions can be observed
Enzymatic activity of a protein can be assayed after separation
Resolution generally not as high as that achieved with SDS PAGE
What is denaturing PAGE ?
Disrupts 2°, 3°, and 4°structures – unfolds the protein
Use of sodium dodeycl sulphate (SDS) confers -ve charge to the proteins
Mobility depends on the size of protein - good for molecular weight determination
Higher resolution than non-denaturing PAGE
Determines relative abundance of major proteins in a sample
Protein-protein interactions or aggregations that occur in vivo won’t be observed
How can you improve resolution ?
Discontinuous systems - separating gel or stacking gel
Proteins are compressed and stacked in order of mobility
Stacking gel contains chloride ions while the electrophoresis buffer contains glycine ions
Leading ions - migrate through gel faster than protein sample
Trailing ions - slower than protein sample
Protein molecules are trapped in a sharp band between these ions
What is Isoelectric Focusing (IEF) ?
Technique for separating molecules based on their different isoelectric points
At pH below the pI, protein will have + charge
At pH above the pl, protein will have a -ve charge
What are IEF gels ?
Strips or columns of
polyacrylamide containing a pH gradient (IPG strip)
Proteins migrate according to charge until they reach portion of the strip where the pH corresponds to their pI
Net charge is 0 and migration stops
Native or denaturing conditions are both possible
Describe the combination of IEF with PAGE:
Combination of IEF with PAGE - 2D Electrophoresis
In the first dimension, proteins are separated based on their isoelectric point (pI), which is the pH at which a protein has no net charge by IEF
In the second dimension proteins are separated based on molecular weight by SDS-PAGE
ID by mass spectrometry
Used to separate complex mixtures
What are the advantages and disadvantages of 2D Gels;
Advantages;
- can resolve proteins that have undergone some form of post translational modification
- different gradient gels available for better resolution
- relatively inexpensive
Disadvantages;
- automated spot picking robots for MS/MS identification are very expensive
- can’t resolve very large proteins, very hydrophilic or very hydrophobic proteins
What is capillary electrophoresis ?
Used for separation of a variety of analytes of different sizes and charges - organic, inorganic ions, metal ions, drugs
Electrophoresis carried out in capillaries
Usually fused silica coated with polyimide
Use of a support medium (gel) is not necessary
Analysis carried out in free aqueous solution
Describe how capillary electrophoresis works:
Samples migrate along the tube due to;
Electrophoreticmigration (EM) - charged molecules move to oppositely charged electrode
Electroendosmosis (E) - Phenomenon due to presence of charged groups on surface of the support, a much stronger force than EM
All molecules move from the anode (+) towards the cathode (-) and are separated on basis of size and charge simultaneously