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How are TLC molecules separated?
Basis of their differences in polarity
How do polar molecules travel?
More polar molecules associate more strongly with the polar stationary phase and so travel much less than molecules of lower polarity which spend more time associated with the mobile phase and so travel further
TLC stationary phase
Silicone dioxide (silica) or aluminium oxide (alumina) attached to a glass metal or plastic sheet
What do hydroxyl (-OH) groups do to stationary phase?
Make stationary phase very polar promoting polar interactions (e.g. hydrogen bonds, van der Waals and dipole dipole interactions) with suitable compounds
Mobile phase in TLC
Many different ones - dependent on the polarity of the compounds being separated
What does separation in TLC depend on?
How much time a compound spends associated with the solvent vs the stationary phase
How does a polar solute act?
High affinity so adsorbed to silica
How does non-polar solute act?
Weak interaction with silica so associates with mobile phase
How does separation of solutes happen?
Differences in the partitioning between the stationary and mobile phases
What stains are used to visualise spots?
Fluorescence - spots appear dark against bright background under UV light
Ninhydrin - reacts with amino acids to give coloured compound
Oxidisers (e.g. permanganate) - oxidation gives brown/yellow spots
How to calculate Rf value?
Distance substance travelled / distance solvent travelled
Rf = 0.00
Great affinity for the stationary phase
Rf = 1.00
Great affinity for the mobile phase
Gel electrophoresis
Separates macromolecules (usually Nucleic acids or proteins) on the basis of their ability to migrate through a gel matrix under the influence of an electrical field
How does separation happen in electrophoresis?
Largely due to mass and charge
What factors affect electrophoretic mobility?
Net charge, size, shape, electrical field strength
How does net charge affect electrophoretic mobility?
negatively charged (anions) move towards the anode (+) and positively charged molecules (cations) move towards the cathode (-)
Highly charged molecules will move faster towards the electrode of opposite charge than do molecules of lesser charge
How does size affect electrophoretic mobility?
Due to frictional resistance small molecules move through the gel faster than larger ones
How does shape affect electrophoretic mobility?
Friction also means migration is affected by shape e.g. globular proteins migrate faster than fibrous proteins and circular DNA migrates faster than linear DNA of the same size
How does electrical field strength affect electrophoretic mobility?
The speed of migration increases with increasing electrical potential (voltage) but increasing voltage too much can cause heating and artefacts
What 2 types of materials typically used to prepare gels?
Agarose and polyacrylamide
Agarose features
Used for Nucleic acids and other very large molecules (>200kDa)
Large pore size
Processed faster
Resolution is inferior
Polyacrylamide features
Used for separation of proteins and small Nucleic acids
Single concentration / gradient gels
Resolution is superior
How is DNA visualised in gel elctrophoresis?
UV transilluminator
What is added to the gel for UV visualisation at the end of the run?
Gel red
Where are the wells placed in tank?
Next to the cathode (negative), and submerged in buffer → towards anodes
How long does loading dye travel?
Migrated about 60% of the way along the gel
What separates in polyacrylamide gel electrophoresis?
Separate proteins in a polyacrylamide gel
What do sodium dodecyl sulphate (SDS) do in polyacrylamide gel electrophoresis?
Binds to proteins giving an even charge density so separation due to differences in mass
Where is sample introduced in polyacrylamide gel electrophoresis?
Into well at top of the vertical gel and electrical field applied
Proteins migrate with smaller proteins migrating further
How are protein bands visualised in polyacrylamide gel electrophoresis?
Staining and compared to molecular weight markers
What is discontinuous system?
Used with a ‘stacking gel’ on top of the main resolving gel (increases resolution)
What are the types of blotting of gels?
Southern, northern, western
Southern blotting
Detection of specific DNA fragments from complex mixtures
Separated by electrophoresis and then transferred (blotted) onto nitrocellulose or nylon membrane
Membrane is then incubated with labelled (radioactive or non-radioactive) complimentary DNA probes which bind to specific DNA sequence of interest. The bound probes are detected e.g. x-ray film
Northern blotting
Detection of specific RNA fragments from complex mixtures
Similar to Southern blotting but RNA is first denatured prior to electrophoresis so mobility is proportional to size. After blotting the blot is probed with complimentary RNA or DNA probes
Southern blotting apparatus
The gel is sandwiched between a filter paper wick dipping in buffer and the membrane. On top are filter papers and absorbent paper under a weight. Buffer is drawn up from the bottom and soaks into the paper above the gel. As it does so it draws the DNA out of the gel and onto the membrane
Western blotting
Detection of specific proteins from complex mixtures
SDS PAGE shows protein bands at particular molecular weights but does not identify the proteins. Western blotting can be used to identify specific proteins in a PAGE gel.
• After electrophoresis the proteins are transferred to nitrocellulose membrane and free binding sites are blocked with a protein solution. The blot is then probed with a (primary) antibody specific to the protein of interest
Detection in western blotting
After ‘blocking’, the blot is probed with a primary antibody (Ab) against the protein of interest followed by an appropriate (correct species and Ig subclass specific) secondary Ab which recognises the primary Ab and is labelled with an enzyme (e.g. HRP). Incubation with a substrate leads to a coloured or chemiluminescent signal to visualise any positive bands