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 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 is currently solubilized and stable in a detergent can be run through the Selector kit to identify additional detergents for downstream applications. This includes all classes of membrane proteins (transporters, enzymes, channels, etc.) expressed in any system.
Detergents may be thawed at room temperate or with gentle warming and mixing up to 50 degrees Celsius, for short durations
The minimum amount of protein required for this assay will be dependent on the sensitivity of your detection method. Buffer, wash and resin volumes can be adjusted throughout the suggested protocol to further miniaturize the protocol and generate more concentrated samples for detection.
Any affinity resin that has been shown to bind the protein of interest, and can be eluted from the resin is compatible. Resins that covalently bind to the protein (i.e. IgG resin), where the protein needs to be removed by proteolysis of the affinity tag are not compatible.
Due to absorbance of some of the detergents in the panel in this range, using A280 absorbance to quantify your protein is generally not recommended for complete detergent sampling. If the user is content to miss the Anapoe-series of detergents from the kit, then this is an acceptable method. Please contact Microlytic Support for additional details on the absorbance behaviors of the detergent panel. Additionally, the frequent use of Histidine-tagged proteins and their elution with Imidazole will also affect absorbance measurements in this range. If your protein has a bound cofactor, such as heme or flavin, absorbance at those wavelengths can be used.
This can be caused by poor binding of the protein to the affinity resin, little elution from the affinity resin, or severe aggregation of your protein sample in all of the detergents. The first step would be to check if protein was eluted from the affinity resin (Step 2) by running a gel (or performing a western blot) of samples from a few wells of the elution plate. If there is no protein present, this is an indication that there was poor binding to the affinity resin, or the protein did not elute. A sample of the affinity resin from the 0.22 um filter plate can be run on a gel to see if protein is still bound.
One way to narrow down the list of promising detergents is to perform small scale detergent exchanges followed by further biophysical analysis. We recommend the use of size exclusion chromatography to ensure the new protein-detergent complex is non-aggregating and monodisperse. Other analyses include dynamic light scattering, thermal stability assays (i.e. CPM), or analytical SEC.
All of the 94 detergents in the panel are available from Anatrace and the catalog numbers of each one are listed in the user manual.
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.