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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.


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)))

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 Selector: A rapid assay for detergent compatibility

 

Selector is a rapid, high throughput assay that explores detergent space to identify those best suited to stabilize the membrane protein of interest in advance of downstream protein crystallization trials.  The assay uses differential filtration in 94 different detergents to generate information regarding both the stability and the size of the protein-detergent complex. The steps of this assay are simple: First, the protein is bound to an affinity resin, washed and eluted in a new detergent.  The eluate is then passed through two filter plates with differing molecular weight cut-offs (MWCO). . The filtration profiles provide stability and relative sizing information that allows the selection of detergents most likely to stabilize a protein in a manner that would be commensurate with crystallization and structure determination.Why should I explore such a variety of detergents?

Integral membrane proteins present a unique challenge to protein chemists and structural biologists. In their natural environment, these proteins exist within a mosaic lipid bilayer that is a dynamic and complex environment. Prior to structural study, however, these must be extracted from the cell membrane and solubilized in such a way as to satisfy the hydrophobic nature of the transmembrane regions while ensuring that hydrophilic domains are in contact with an aqueous phase.  While a significant number of detergents may be capable of keeping the protein in a soluble form, the stability, homogeneity and activity of any given membrane protein can be tremendously affected by the properties of the solubilizing component.
 

Deposits to the Protein Data Bank for membrane proteins comprise broad range of detergent and detergent-lipid complexes as part of the protein preparations leading the diffraction-quality crystals.  Thus, while certain detergents (DM, DDM or OG, for example) are known for their success in stabilizing integral membrane proteins for structural studies and are reported at high frequency, significant effort would be required to explore detergent space, even to cover detergents previously successful for protein crystallization.
 

What does this kit contain?

1 x detergent screening plate containing 150 µl of 94 detergents at 2x working concentration, one blank and one position for the detergent currently stabilizing the protein sample to be screened

1 x 0.22 µm filter plate with receptacle plate for detergent exchange

1 x receptacle plate into which the proteins and newly exchanged detergents will be eluted

1 x 300  MWCO filter plate with matched receptacle plate

1 x 100 MWCO filter plate with matched receptacle plate.
 




Analytic Crystallizer:

The Analytic Crystallizer kit comprises a two-step method for optimal crystallization screening of detergent-solubilized integral membrane proteins (IMPs) in a manner independent of detergent identity. The kit includes a solubility prescreen (Optimizer) that is used to determine the appropriate protein concentration for use with the crystallization screen, which is then followed by the 96-condition crystallization screen (Crystallizer). The use of Optimizer allows the experimenter to reduce the amount of crystallization conditions to be screened in the first screening pass and to increase the likelihood of crystallization success. The result of coupling the Optimizer and Crystallizer solutions sets is the assurance that the majority of the crystallization screen solutions will result in supersaturation of the protein without excessive non-crystalline precipitation.

 

The conditions for Crystallizer have been formulated to avoid redundancy and maximize coverage of crystallization space. Precipitants and salts were mined from the conditions most frequently used in successful membrane protein crystallization experiments, as available in the Membrane Protein Data Bank. Also sampled are six buffers ranging from pH 4.5 to 9.5 and 18 distinct salts.

 

The formulations provided here have been further tuned to render the screen detergent-independent, meaning that the precipitant and salt concentrations have been selected to minimize phase separation that typically occurs for solutions containing high concentrations of detergents that are typically used to maintain the solubility of membrane proteins. 




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