Describe the purification of proteins starting from the body and its isolation.
1) Homogenization
- >it is the crushing, grinding or blending the tissue of interest into an evenly mixed solution
2) Centrifugation
- >can then isolate proteins from much smaller molecules before other isolation techniques are employed
-the next most common isolation techniques are chromatography and or electrophoresis
Describe the process of electrophoresis. Also describe from where to where these compounds travel to.
- works by subjecting the compound to an electric field
- > which then the compounds move according to charge and size
- note negatively charged compounds will migrate towards the positively charged anode
- > positively charged compounds will migrate towards the negatively charged cathode
What is the velocity of migration referred to as of a molecule going through electrophoresis?
- it is the migration velocity
- directly proportional to electric field strength and the net charge of the molecule
What kind of gel is used for protein electrophoresis?
- polyacrylamide gel is used for protein electrophoresis
- > proteins travel through this matrix in relation to their size and charge
- > smaller particles and highly charged molecules move faster through this electric field
Describe the process of Native Page.
-it is a method for analyzing proteins in their native states
What are the limitation of native page? What is it useful for?
- it is limited by varying mass to size ratios and mass to charge ratios
- > proteins migrate due to mass to charge ratios
- > different proteins may experience the same level of migration due to these properties
-it is useful to compare the molecular size or charge of the proteins known to be similar in size from other methods
Describe the characteristics of SDS-Page? What factors of a compound are taken into account during SDS-Page?
- it is a polyacrylamide gel electropheresis tool
- separates proteins based molecular mass alone
- > add SDS to denature proteins(disrupts noncovalent interactions)
- > SDS binds to proteins and creates large chains with net negative charges
-only the electric field is taken into account and the frictional coefficient(depends on mass) when a protein moves through a SDS-Page gel
What is the isoelectric point
- it is the point at which the protein or amino acid is electrically neutral
- > so that means equal number of positive and negative charges
- > for individual amino acids, this is referred to as a zwitterion
Describe the process of isoelectric focusing
- it exploits the acidic and basic properties of amino acids
- > separates based on isoelectric point
- > there is an acidic gel at the anode
- > there is a basic gel at the cathode
- > the center is neutral
What is the key concept of chromatography
- the more similar the compound is to its surroundings
- >the more it will stick and move slowly through its surroundings
When is chromatography preferred over electropherisis?
-it is preferred when large amounts of protein are being separated
Describe the process of chromatography
1)place a sample onto the stationary phase or the adsorbent
2) Run the mobile phase through the stationary phase
- >this allows for elution of the compound through the stationary phase or for it to run through(elute just means for the sample to run through the phase)
- >those that have high affinity for the stationary phase will barely migrate at all
- >those that have high affinity for the mobile phase will run much faster
What is meant by the term retention time?
-the amount of time a compound spends in the stationary phase
Describe the process of column chromatography
- column is filled with silica or alumina beads as an adsorbent
- gravity moves the solvent and compounds down the solvent
- eventually the solvent drips out of the end of the column
- > different fractions that leave the column are collected over time
What factors determine how fast a compound moves during column chromatography
1) size
2) polarity
- >less polarity= faster elution
Why use column chromatography
- because it can be used to separate and collect macromolecules other that proteins
- > eg; nucleic acids
Describe ion-exchange chromatography
- a column is coated with a charged substance
- > they attract or bind to compounds of the opposite charge
- a charged protein is put through the column
- > the column attracts proteins of the opposite charge
- > the oppositely charged proteins will have a longer retention time
Describe size-exclusion chromatography
- it contains beads in the column containing tiny pores of varying sizes
- > these tiny pores allow for small compounds to enter, thus slowing them down
- > larger compounds can’t fit into these pores and have to go around the pores
- > so they travel faster
Describe affinity chromatography
- columns bind to any protein of interest by creating a column with high affinity for that protein
- > eg; coating the column with receptors that bind to the protein or a specific antibody for that protein
- once the protein is retained on the column
- > it can be washed out with a free receptor(target/antibody)
- > this eluent will compete with the bead-bound receptor and ultimately free the protein from the column
-note eluents can be created with a specific pH or salinity level that disrupts the bonds between ligand and protein of interest
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Chapter 2.18
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Chapter 2.27
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Chapter 2.36
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Chapter 2.41
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Chapter 2.57
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Chapter 3.117
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Chapter 3.25
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Chapter 3.319
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Chapter 4.17
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Chapter 4.25
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Chapter 4.33
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Chapter 4.413
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Chapter 5.111
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Chapter 5.217
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Chapter 6.110
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Chapter 6.28
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Chapter 6.310
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Chapter 7.111
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Chapter 10.43
