•Solubilisationof proteins is done with a detergents concentration above the CMC to ensure the incorporation of membrane lipid into detergent micelles.
•CMC = critical micelle concentration
depends on temperature, ionic strength and pH of the buffer and concentration of uncharged substances like urea or alcohol

Some detergents
•Ionic detergents:
Sodium-Dodecylsulfate:denatures Proteins (􀃆SDS-PAGE)
Na-Deoxycholate:     preticipatesby pH<6.8
                                  preticipatewith Ca²⁺or Mg²⁺
In General: No ionic Detergents in purifications with depend on the charge of the proteins.
•Non-ionic detergents:
Triton X-100             Phenyl groups 􀃆strong Absorbance at 280 nm
Tween 20                   like Triton X-100 not dialyzable
•Zwitter-ionic detergents
Chaps                         dialyzable

Which proteins are purified?
•Metabolic pathways
•Energy production
􀃆Aim: biochemical characterisation (Reactivity, subunit composition, organic and inorganic cofactors, 3D structure)
…and why?

Purification strategies
•Protein stabilisation:
–Integral membrane Proteins: Solubilisation
–Purification at 4°C: reduces protease activity
–Addition of protease inhibitors: commonly used are EDTA and PMSF (toxic)
–Quickly load on first column after cell disruption and ultra centrifuge
•Main impurities are removed first, lesser in the second or thirdstep
•Max 5% impurities are acceptable
In general:Max 4 purification steps
No steps with purification factor < 5
No steps with < 30% yield
No steps which last longer than one day and one night

Chromatography
•Separation material in columns, streamed by buffer
→liquid chromatography
HPLC: high pressure/performance liquid chromatography
FPLC: fast protein liquid chromatography

FPLC unit

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