BCHM 218 Lecture Notes - Lecture 7: Column Chromatography, Protein Purification, Shear Force
5 May 2018
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BCHEM 218 week 3 lecture 1
Protein
purification
Isolation and purification of protein
Cell lysis:
- Detergent: compromises integrity of cell memb
- Shear force: physically disrupted by applying high f sonic waves or shaking sample
- Low ionic salt: treatment of cell cultures w low ionic salt= water absorption pop
P change: high P=lysis
Centrifugation: repeated centrifugation cycles at diff rotational speeds, radial acceleration causes denser
particles to sediment at btm (pellet) less dense to rise (supernatant)
Fractionation: column chromatography used to sep protein
from cell lysate, mix dissolved in fluid then passed thru
structure (stationary phase)
Protein mix applied to column containing resin or matrix that
interacts diff w diff proteins
Buffer causes bound proteins to dissociate from matrix
(elution) at diff times
Eluted proteins: collected in fraction collector which gradually
moves test tubes under column keeping the proteins sep
Types of
column
chromatograph
y
ion-exclusion: resin contains an anion exchange resin (+vely
charged) and binds anions or a cation exchange resin (-vely
charged) and binds cation
- Rate of release depends on nature of charged aa on
surface
size-exclusion: column matrix contains beads that have pore specific size allowing smaller proteins to enter,
larger proteins pass easier and faster
Affinity: many proteins specifically bind other molecules or ligands, column contains ligand covalently attached
to matrix, proteins can be end to facilitate affinity by adding aa to being or end
Ex. GST tag promotes greater expression and solubility of recombinant proteins and can binds GSH (specific sub
for GST, used to capture GST tagged proteins)
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