Lectures 14, 16 Flashcards
How do you purify by solubility, ionic charge, polarity, size, and binding specificity?
Solubility- salting out
Ionic charge- ion exchange chromatography
Electrophoresis
Isoelectric focusing
Polarity- reverse phase & hydrophobic chromatography
Size- dialysis
Size-exclusion chromatography
Gel electrophoresis
Binding specificity- affinity chromatography
What is centrifugation?
What is a supernatant?
Initial purification
If the protein is soluble it can be partially purified by removing larger/denser contaminants by differential centrifugation
The supernatant is the liquid at the top of the centrifuge tube
Slide 4 L14
What is salting out?
Common and reversible method
As [Salt] Increases, the + and - ions will compete with the hydrophilic surface amino acids for water
The protein molecules with insufficient hydration will aggregate and lose solubility
Slide 7 L14
What is dialysis (de-salting)?
Excess salt must be removed as it reduces solubility and can impede subsequent isolation steps
Dialysis removes salt by dialysis tubing that had pores with a specific molecular weight cut off that allow smaller molecules (salt) to pass
Slide 8 L14
What is chromatography?
What is stationary and mobile phase?
Techniques to separate mixtures based on physical properties such as size or charge
Stationary phase- a substance that the compounds to be separated pass by or interact with
Mobile phase- the carrier for the compounds to be separated
Different samples can have different affinities for the stationary and mobile phase, this means different rates of migration and separation of the molecules
What are the 5 steps of column chromatography?
- Pouring- glass tube with valve at bottom is filled with a mixture of stationary and mobile phases
- Packing- opening stopper and letting some liquid run out to make the stationary phase more densely/evenly packed
- Loading- sample of interest is placed on top of stationary phase
- Running/eluting- sample flows into the column and more mobile phase is added on top to keep the column from drying out
- Collecting- samples of defined volumes are caught into tubes at the bottom and saved/tested
What is size exclusion chromatography?
What are the important volumes?
Porous beads make up the stationary phase
Large proteins cannot enter the beads so they migrate around them (just Vo)
Moderate proteins enter beads sometimes which increases the volume available and move more slowly (Vo and some Vi)
Small proteins do not interact with the beads and move the slowest (just Vt)
Volume of column is Vt (πr^2 x h)
Volume outside the beads is Vo
Volume inside the beads is Vi, Vt x 0.7= Vi
Volume at which a sample is eluted is Ve
Slide 12-13 L14
What is the partition coefficient?
Fraction of the volume of the column available to the sample
Kav = (Ve-Vo) / (Vt-Vo)
Slide 14 L14
Small to large protein examples
Slide 15-17 L14
How is the partition coefficient used to estimate molecular weight?
Slide 19 L14
What is the range of Kav where we get poor separation fo proteins?
What causes the dilution?
0.8
What is ion exchange chromatography?
Column of positive or negative charged beads forms the stationary phase Anion exchange (column is +, targets are -) Cation exchange (column is -, targets are +)
Proteins in solution get carried down by gravity and will get slowed by the beads proportionally based on charge
Specific combo of mobile and stationary phases is chosen to retain protein of interest (pH)
What is ion exchange chromatography?
Slide 3-5 L16
Like charges or neutral molecules will elute quickly
The greater the charge on a protein, the slower it will travel down the column
Sample are collected and each can be tested for the protein of interest
Can alter pH to reduce the charge of the proteins bound to the column or increase salt concentration to increase competition for binding the stationary phase
What is affinity chromatography?
Slide 6-7 L16 Specific trap for protein of interest attached to stationary phase Enzyme -> substrate Receptor -> enzyme Antigen -> antibody His protein -> Ni
Elation can be problematic
High purity can be achieved
What is the order of specificity of chromatography methods?
Which are easiest to do?
- Affinity chromatography (hardest to do)
- Ion exchange chromatography
- Size exclusion chromatography (easiest to do, least specific)
What is electrophoresis?
Slide 9-10 L16
Migration of ions in an electric field
Velocity is directly proportional to charge and inversely proportional to size and shape
Proteins have I here t charge differences and can also be coated with charged particles
Gel slows down movement and prevents diffusion when power is turned off
Opposite charges attract (size matters too, bigger sizes have more friction)
