Analytic Extractor:
Integral membrane proteins (IMPs) insert into biological membranes such that the cell membrane, which is a lipid bilayer approximately 30 Å in thickness, shields the hydrophobic membrane-spanning regions of the protein. To purify IMPs, the protein must first be extracted from the bilayer through the replacement of cell lipids with solubilizing detergents. The selection of detergent for both the initial solubilization of the IMP and for downstream study is exceptionally important, as one must ensure that the selected detergent does not inhibit function, cause irreversible denaturation or interfere with protein purification or crystallization.
Previous studies on the selection of detergents for the initial extraction of IMPs have demonstrated the importance of detergent screening (REFS). One of the most exhaustive studies was performed by White et Al. (2007) in which 122 high-expressing predicted membrane proteins were screened in 6 commonly used detergents Triton X-100, LDAO, FC-12, C8E4 and DDM. In these tests, the zwitterionic detergetns LDAO and FC-12 were the most efficient at solubilizing yeast membrane proteins; however, all of the detergents in the panel solubilized at least 35% of the proteins tested.
The Analytic Extractor kit contains a panel of eight detergents commonly used for the solubilization of IMPs from the cell membrane. The detergents in this panel were chosen based on their prior effectiveness and use in the literature, and the working concentration of each detergent is dependent on the critical micelle concentration (CMC) of each detergent (i.e. higher concentrations of lower CMC detergents compared to higher CMC detergents) (Ref to Wiener, 2004). The results from this assay will aid in the selection of a detergent for further downstream studies, including x-ray crystallography, NMR, and biochemical assays.
|
Common Name
|
Full Name
|
Anatrace Catalogue #
|
CMC (% (w/v)))
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Provided Concentration (5x stock)
|
Working concentration
|
|
Anapoe-100
|
Triton X-100
|
APX-100
|
0.015%
|
5%
|
1%
|
|
C8E4
|
Tetraethylene glycol monooctyl ether
|
T350
|
0.25%
|
10%
|
2%
|
|
C12E8
|
Octaethylene glycol monododecyl ether
|
O330
|
0.0048%
|
2.5%
|
0.5%
|
|
DM
|
n-Decyl-β-D-maltopyranoside
|
D322
|
0.087%
|
5%
|
1%
|
|
DDM
|
n-Dodecyl-β-D-maltopyranoside
|
D310
|
0.0087%
|
5%
|
1%
|
|
FC-12
|
FOS-CHOLINE®-12
|
F308
|
0.047%
|
5%
|
1%
|
|
LDAO
|
n-Dodecyl-N,N-dimethylamine-N-oxide
|
D360
|
0.023%
|
5%
|
1%
|
|
OG
|
n-Octyl-β-D-glucopyranoside
|
O311
|
0.53%
|
10%
|
2%
|
It is important to note that regardless of the success of a particular detergent in extracting the IMP from the cell membrane, subsequent experiments may require the reduction, removal or exchange of the detergent. For assistance in selecting optimal detergents for downstream experiments, please refer to the Selector kit within the Analytic line.
Analytic Extractor is a quick start kit that contains detergents commonly used for extraction and/or crystallization of Integral Membrane Proteins (IMPs) from the membranes of both prokaryotic and eukaryotic cells.
Analytic Selector provides a rapid assay for the identification of detergents that maintain the solubility of the integral membrane protein, minimizing aggregation and promoting stability.
Analytic Crystallizer is a unique two-step protocol for membrane protein crystallization, designed to help you reduce the amount of conditions to be screened in the first pass and increase the likelihood of crystallization success.
Any membrane protein that has quantifiable expression can be run through the Extractor kit to identify detergents suitable for solubilization. This includes all classes of membrane proteins (transporters, enzymes, channels, etc.) expressed in any system.
Scaling up protein preps is a common, and frustrating, problem with membrane protein purification. When moving to larger preps, some solubilization parameters, such as detergent concentration and solubilization time may have to be varied.
We provided the most commonly used detergents for membrane protein extraction in this kit; however, there could be an instance where the membrane protein is recalcitrant to solubilization. As a positive control, you can try to attempt solubilization in a harsh detergent like SDS to see if extraction of the protein is possible. Additionally, changing the solubilization parameters, including the buffer, solubilization time, and solubilization temperature may be necessary. Lastly, if working on eukaryotic membrane proteins, the addition of cholesterol hemisuccinate may be often necessary.
Analytic Extractor is a quick start kit that contains detergents commonly used for extraction and/or crystallization of Integral Membrane Proteins (IMPs) from the membranes of both prokaryotic and eukaryotic cells.
Analytic Selector provides a rapid assay for the identification of detergents that maintain the solubility of the integral membrane protein, minimizing aggregation and promoting stability.
Analytic Crystallizer is a unique two-step protocol for membrane protein crystallization, designed to help you reduce the amount of conditions to be screened in the first pass and increase the likelihood of crystallization success.