Lecture 3 Flashcards
How do cells protect themselves from oxidative stress
They have a reducing cytosolic (inside) environment and the outside is oxidizing
This protects themselves from oxidative damage
Ex. If protien needs disulfide binds, that won’t form properly inside the cell since it would be reduced inside the cell. So the cell excretes it.
What is ion exchange chromatography
A way to separate charged molecules like protiens nucleic acids or ion based on their charges and pl
There anion exchange: resin is postive charge, catches anion
Cation: resin is negatively charged, catches cations
In ion exchange chromatography what is used to elulte the protien
What does this mean
A low to high salt gradient
This means that before doing lEC you need to start with low salt (so reduce the salt concentration)
What method is used before ion exchange chromatography
How does it work
Dialysis
The salt is originally concentrated with the solution of the protien
The dialysis bag in water has a semipermeable membrane so that salt can leave the solution in the bag
Salt leaves solution and reaches equilibrium
What is size exclusion chromatography (gel filtration)
Separates molecules based on size
The buffer composition doesn’t affect the separation between molecules, just the size
Bigger molecules elute faster since they dodge the gel beads by smaller go slower since they go through the beads
What is an aggregate/void peak
The largest molecules that elute out of gel filtration first are usually aggregated protien that we don’t want
When this comes out it’s called a aggregate/void peak
What is a bad sec result
You get bad separation of peaks, have contaminant peaks
The signals of the pure protein and the contaminants merge and aren’t distinguishable
What is 6xHis calmodulin
What does fusion mean
A soluble cytosolic protein that has a 6 his tag
Has a protease cleavage site (TEV)
When calcium binds to it the nonpolar (hydrophobic) residues are exposed
If whole MW given with (fusion) that means that the entire weight with the his tag
What is the first step to purifying 6xhis CAM
How do you do it
In any step you first need to lyse the cell to obtain the protein
Can lyse by chemical, sonication, or French press methods
What is a lysis buffer
What’s in it and why
After lysing the cell you put the pelleted (lysed) cells in it
Made of:
DNase1: helps breakdown dna so the solution doesn’t get too dense and not purify properly
Lysozyme: breaks down peptidoglycan, gram + bacteria have thick peptidoglycan, ecoli doesnt have lysozyme so adding it helps the breakdown of the cell
low imidazole concentration : some protiens in the lysed cell naturally have histidine, don’t want them to bind to the column so you add imidazole so they stay in buffer solution and you get rid of them
protease inhibitor or a cocktail of inhibitors
Glycerol (mimics dense cellular environment by making solution viscous)
What do you add in the lysis buffer if your protein of interest binds dna/rna or is highly positively charged
Add mgcl2 or cacl2
And benzonase (enzyme that degrades RNA/DNA)
After the cell has been lysed and its contents are in the buffer what is step 2 of purification
IMAC (to purify the lysate)
The filtered super area from step 1 is placed onto nickel resin coated with the IMAC wash buffer
In step 2 of purification what is in the iMac wash buffer and why
How does the buffer change
Low imidazole so that his tagged protien we want binds to the IMAC instead of imidazole (rest of contaminants gets eluted)
Then elute again on IMAC with same buffer but higher imidazole concentration to get 6xhis CAM off column and eluted
If our protein needed to be reduced (had cystines) what would we do
After eluting it with IMAC use TSEP as a reducing agent in all buffers afterward.
Not BME before doing IMAC because it binds to the nickel column
What is step 3 to purifying 6xhis CAM
Size exclusion chromatography
