Protein Purification Methods Flashcards
What’s the average length of proteins? What’s the size range?
Average= about 400 residues
Protein size range= about 30-35,000 amino acids.
How can one obtain multiple milligrams of pure protein? (Hint: 2 ways)
1 obtain protein from natural sources.
2 Recombinant expression: clone and overexpress from one species in a rapidly growing cell of another species: “heterologous expression”.
3 Amino acid synthesis. (Only works for small protiens)
Define fractionating.
To separate specific protein of interest.
Means you are dividing your protein into fractions that contain similar properties within each fraction.
How do you fractionate specifically solubility in proteins?
Procedure: Salting out.
Often called ammonium sulfate fractions .
How do you fractionate specifically size in proteins?
Two options.
1. Selective dialysis: dialysis through a size selective membrane.
2. Gel filtration chromatography.: percolation through a porous solid matrix.
How do you fractionate specifically charge in proteins?
Ion exchange chromatography: dependent on the isoelectric point of a protein.
How do you fractionate specifically “binding specificity” in proteins?
Affinity chromatography: based on binding to a known target.
Can you explain how this type of fractionation works?
Explain how selective dialysis works? What is it fractionating?
Define chromatography
A chemistry technique that’s purpose is to separate mixtures.
Explain how gel filtration chromatography works. What is it fractionating?
Note: The beads are named after the complimentary material that’s exchanged. So Weak cation exchanger has an anion on the bead, NOT a cation.
Explain what ion exchange chromatography is dependent on. What is it fractionating?
It sorts based on the isoelectric point.
Define what the isoelectric point is. (pI)
Note: while the isoelectric point of a protein molecule depends primarily on its amino acid composition. The 3-D structure of the protein also comes to play.
Since the pKa of individual functional groups in a folded protein depend also on the environment of the group that is isoeletirc point of a protein depends also on its confirmation.
How do people find exact isoelectric points of complicated 3-D proteins?
By finding the pH where the protein no longer moves in an electric field. This is because neutral proteins have no charge therefore they don’t move in and electric field.
Can you explain and visualize this slide?
Explain how affinity chromatography works. What is it fractionating?
What are 3 methods for determining/measuring protein concentration.
What amino acids does UV spectroscopy use to find concentration. (Hint 3) And what wavelength do these amino acids absorb at?
Explain how staining with coomassis blue determines protein concentration.
Explain how ELISA works in determining SPECIFIC protein concentrations.
Major requirement: Needs primary and secondary antibodies that bind to specific protein for this to work.
