Chapter 5: Affinity Chromatography Flashcards
in affinity chromatography, do the proteins stick to the decorated beads or to the buffer?
they stick to the decorated beads
why does affinity chromatography so often involve genetically encoded affinity tags?
because the target protein may not have a natural affinity to anything you could decorated your beads with.
what does Dr. Shimko focus on the most in the affinity chromatography section?
the genetically encoded affinity tags
why would you ‘tag’ a protein in affinty chromatography?
To facilitate the protein’s affinity for the ligand you decorated your beads with
How is a genetically encoded affinity tag added to a target protein?
You put the DNA for your target protein into a vector that contains the tag. The vector then expresses the target protein-n-tag combo for your experiment.
what are genetically encoded affinity ‘tags’ actually made of?
Amino acids
what is a fusion protein?
what you call a target protein that has had a tag added to it
what are the 3 genetically encoded affinity tags Dr. Shimko talks about?
- polyhistidine
- MBP ( maltose binding protein)
- GST glutathione-S-Transferase
what does it mean to say that a purification process has HIGH activity?
That it yielded you a relatively ‘pure’ final product
It means that your final product has lots of target protein relative to extraneous stuff/contaminants
what does it mean to say that a purification process has LOW activity?
That it yielded you a relatively ‘UNpure’ final product
It means that your final product does not have a lot of target protein relative to extraneous stuff/contaminants
Is purification ‘activity’ the same as product yield?
no
What is the benefit of using large genetically encoded affinity tags?
They can fold and increase target protein stability
How do you remove a genetically encoded affinity tag from a target protein?
Protease enzyme will cleave the tag off your target protein.
do genetically encoded affinity tag impact the function of target proteins?
no
is poly histidine a small genetically encoded affinity tag or a big one?
small. it is only about 6-10 amino acids
