Sepration Techniques Flashcards
What are the ways you can separate substances in biology
Centrifuge, paper and thin layer chromatography, affinity chromatography, gel electrophoresis, isoelectric point
Describe the centrifuge process
-Samples are spun at incredibley high speeds
-This separates substances according to density
-More dense components settle to form the pellet whilst less dense components remain in the supernatant
Describe paper and thin layer chromatography
-Used to separate different substances such as amino acids and sugar
-The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used
Describe affinity chromatography
Used to separate proteins
A solid matrix or gel column is created with specific molecules usually receptors bound to it
Soluble target proteins in a mixture with a high affinity for these molecules become attached to them as the mixture passes down the column
Other non target molecules with a weaker affinity are washed out
Describe gel electrophoresis
Used to separate proteins and nucleus acids
Native gels will separate proteins by the shape and size and charge
They do this by ensuring the do not denature the molecule
Sds-page will separate proteins by size alone
They do this by giving all the molecules an equally negative charge and denatures them
How does gel electrophoresis work
The samples are loaded into wells in a gel which will have an electric current running through it. As they are usually charged molecules they will love towards the opposing charge. Smaller molecules will travel faster then larger molecules so will travel further
Describe isoelectric point
Used to separate proteins from a mixture
The IEP is the specific ph at which a soluble protein as no jet charge and will precipitate out of a solution
If the solution is buffered to a specific ph only the proteins that have IEP of that ph will precipitate
What’s the steps in the ph gradient
1- two different proteins with differing IEPs loaded into gel matrix
2-proteins migrate towards the charges until they reach an area with the ph of their IEP
3-proteins stop migrating as they have no net charge and precipitate out
Why does a protein stop migrating through the gel at its IEP in the ph gradient
Because it has no net charge
What do charged macromolecules move through
An electric field applied to a gel matrix
What can soluble proteins be separated by
Using an electric field and a ph gradient
