Protein PurificationExtraction and Clarification
Solubilisation of membrane proteins
In this case of membrane proteins the extraction process should be followed by a number of different steps:
- removal of unbroken cells from the cell lysate by low speed centrifugation (20 min at 10 000 g).
- isolation of the membrane particles from the supernatant by ultracentrifugation
(60 min at >100 000 g).
- washing of the membrane particle to remove all soluble proteins.
- solubilization of protein from the membrane particles by a detergent.
The extend of the solubilization and the stability of the solubilized protein depend on the detergent type and concentration. It is not possible to give general rules for either of these variables and they have to be optimized experimentally. Important for the solubilization is the detergent-to-protein ratio. At low ratios (1:10) the membranes are lysed and large complexes of are formed containing protein, detergent, and membrane lipids. With progressively larger ratios smaller complexes are obtained. Finally, at ratios of 10:1 to 20:1 individual detergent-protein complexes are formed free of membrane lipids. To determine the optimal conditions it is important to vary both the detergent and the protein concentration.
Commonly used detergents and their critical micel concentrations (cmc) are listed in the table below.
|Class of detergent||examples||cmc (mM)|
|Ionic||sodium deoxycholate||4 (at pH >8)|
- The solubility of membrane proteins is affected by the ionic strength of the solutions. Therefore, 150 mM NaCl is usually included in solubilization buffers.
- Polyols stabilize solubilized membrane proteins. Typical glycerol concentrations range from 5-50% (v/v). High concentrations of glycerol will increase the viscosity of the preparations that can cause problems in the ultracentrifuge and on chromatographic columns.
- The buffer concentration can affect the solubility and stability of proteins. Especially phosphate buffers are known for their solubilizing properties. Concentrations of 0.1-0.5 M phosphate buffer increase the solubility of membrane proteins.
- The presence of detergent will have a significant effect on subsequent chromatography steps.