Semester 2 Flashcards
How does gel filtration work?
- molecules in solution separated by size as passed though column packed w/ gel matrix
- matrix consists of porous beads
- smaller molecules can diffuse further into pores of beads
- larger molecules can’t enter many pores (if any)
- so largest eluted first and smallest last
- the smaller the molecule, the more of the total vol of column it passes through, so greater the vol of eluent req to elute it
What is the vol of eluent proportional to in gel filtration?
- time taken for collection
What is the result of using small beads in gel filtration matrix?
- less diffusion, so elution peaks sharper and separation better?
What is void volume (Vo)?
- molecules excluded from beads elution vol, equal to liquid outside beads
What is elution volume (Ve)?
- molecules of diff sizes which can enter bead are eluted w/ Ve
What can affect elution vol (Ve), and what does this mean?
- flow rate
- buffer composition
- temp
- so calibration procedure and experiment should be carried out under same conditions
What is whole vol of liquid in column (Vl)?
- elution vol of smallest molecules which can completely penetrate beads?
How is vol of liquid inside beads (Vi) calc?
- Vl - Vo
How is total column vol (Vt) calc?
- Vo + Vi + Vs
What is vol of solid beads (Vs) and what is its normal value?
- varies for diff types of gel filtration matrices and falls in range between 5% and 15% of total column vol?
What are some of the practical uses of gel filtration?
- desalting or buffer exchange
- purification of macromolecules
- analysis of oligomeric state of protein and protein complexes (what we did)
What gel filtration matrix did we use, and what separation range did this provide?
- Sephadex G100 superfine
- separating range = 4000Da to 100,000 Da
What improvement was dev to Sephradex matrix, and what does it involve?
- Superdex
- cross linked agarose beads w/ large pores filled w/ dextran dictating final pore size
- adv is high res w/ short run times and good recovery
How are calibration plots made for gel filtration experiments?
- plot elution vol versus size
- in practise use molecular weight as measure of size of molecules
- but linear relationship between elution vol (Ve) and logMW
- most commonly do Kav vs logMW
What is a Kav value, and how are they calc?
- proportion of vol inside beads available for a given vol
- (Ve - Vo) / (Vt - Vo)
Does Sephradex have good separation power?
- no, relatively poor
What is important during a gel filtration experiment?
- columns must be kept vertical
- avoid disturbances at top of column during sample app
- don’t touch frit when applying sample
- do not change positions of any components and don’t let column run dry
What can gel filtration be used for?
- purify proteins
- determine molecular mass and subunit composition of oligomeric proteins
What is SDS-PAGE used for?
- analyse extent of purity of protein sample and to estimate molecular size of proteins in solution
What is affinity chromatography used for?
- powerful way to select for correctly folded proteins
What can be used to monitor efficiency at each step of purification process?
- calc of enzyme activity and its value per mg of protein (specific activity)
What is enzyme activity, and what are its usual units?
- 1 unit of activity is amount of enzyme needed to convert 1 μmole of substrate to product in 1 min
- units/ml
How is specific activity calc, and what are its usual units?
- enz activity / protein conc
- units/mg
How is total activity calc, and what does it provide?
- enzyme activity x vol of sample
- provides total no. units in sample at this stage