Question

Fundamentals of Biochemistry Tutorial: Protein Analysis Techniques

Answer 1

\({\bf{Protein~Separation:}}\)
- typically based on charge, size, or binding properties
- sourced from tissue/microbes

Basic Procedure:
1. break open the cell/tissue and separate protein into crude extract
2. separate into fractions based on shared properties, fractionation.
ex: salting out, which extracts proteins based on solubility in salt
ex: dialysis, which separates proteins based on size
ex: column chromatography, which separates based on reactivity/charge, etc.
ex: ion-exchange chromatogarphy, which separates based on charge
- anionic groups (cation exchangers) + cationic groups (anion exchangers) studded in a column, binding ability of proteins depends on pH
3. re-collect the fractions and test for desired property

for column chromatography, resolution increases w/ column length, but diffusion rate decreases, which can cause diffusional spreading and interfere w/ resolution

Answer 2

\({\bf{Other~Types~of~Chromatography:}}\)
- size-exclusion chromatography/ gel filtration: physically filters proteins based on size
- affinity chromatography: column contains ligands, proteins are separated based on binding ability
- HLPC: uses high pressure pumps to increase the rate of protein flow down the column

Answer 3

\({\bf{Electrophoresis:}}\)
- based on movement of charged proteins in E-field
- polyacrylamide gels slow movement of proteins proportionally to charge/mass ratio
- electrophoretic ability: (mu) = V/E = Z/f
where V is the velocity, E is the magnitude of the electric field, Z is charge, f is frictional coefficient
- sodium dodecyl sulfate: detergent used in electrophoresis
binds to proteins proportionally to mass
- dye added to visualize the locations of the proteins on the gel and determine MW



\({\bf{Isoelectric~Focusing:}}\)
- mix of low MW acids/bases on gel
- protein is applied to gel and migrates until pH = pI

above 2 methods, when combined, are called two-dimensional electrophoresis

Answer 4

\({\bf{Analysis~of~Enzymes:}}\)
- can be separated based on catalytic activity
- enzyme activity: 1.0 unit = amount of enzyme that converts 1.0 micro moles of substrate to product at room T
- total activity = total enzyme units
- specific activity = enzyme per mg protein, also proportional to purity
- both total protein mass and specific activity decrease over time but specific activity decreases much less, yielding purer product with each step of the purification
- purification considered complete when specific activity remains constant per protein

Answer 5

Source material is section 3.3 of Principles of Biochemistry 7th edition by Nelson, David L., and Cox, Michael M.