One of our scientists - Tim Craig - has turned his attention to a fundamental barrier that needs to be overcome in any membrane protein research project: functional extraction of membrane proteins from their biological membrane. Every membrane protein target needs to be matched with a particular detergent reagent or a set of detergents. Since Tim was working with fluorescently labeled membrane proteins, he applied an experimental approach that has gained popularity in recent years: FSEC. That is: Fluorescence-Detection Size-Exclusion Chromatography; this is primarily a pre-crystallization method for membrane proteins that has been developed in Eric Gouaux' lab (see references below).
This is how it works: solubilize a membrane with a particular detergent and apply the resulting sample to size exclusion chromatography with a benign detergent (i.e. dodecyl maltoside) in the mobile phase. Repeat with as many detergents as possible. Inspect the traces for symmetry of the (largest) peak and absence of signal in the void volume. Pick the best detergent(s) to establish a purification and crystallization procedure. The neat trick here is that such crystallization-relevant information can be obtained at a very early stage in your membrane protein project, for instance once you can prepare membranes. This methodology solves the hen-and-egg problem (need to have a detergent in order to be able to purify a particular membrane protein, need to have a pure membrane protein sample in order to test which detergent is compatible) and is simple to automate with standard HPLC instrumentation.
Since preparing many different detergents is a laborious process we thought we'd pack them into a kit. And this is it, the Wizard: TIME screen Use the Wizard: TIME screen to conveniently determine the detergent that successfully extracts your target membrane protein from a membrane preparation.
In order to maximize both, extraction efficiency and membrane protein stabilization, each of the 84 different detergent reagent is formulated at 2% (w/v) together with the co-detergent cholesterolhemisuccinate. Positive and negative controls specifically for FSEC make this screen a very convenient tool for fast, simple and information-rich membrane protein extraction assessment.
The complete listing of detergents in the Wizard: TIME screen are available here.
Our recommended Wizard: TIME screen sample treatment protocol:
1. Mix 125μL of your membrane preparation with the contents of each well in a fresh 96-well plate (membranes should be prepared at a concentration that allows for good signal:noise in the analytical assay of your choice).
2. Incubate the mixture to allow for protein extraction (different incubation times and temperatures will influence extraction efficiency, however typically 1 hour at 4°C can serve as a good starting point for extraction optimization).
3. Separate extracted material from debris by ultracentrifugation at >100,000 x g for 20 minutes using a small volume rotor or by filtration using a filter. Keep either the ultracentrifugation supernatant or the flitration flow through and
4. Analyze samples with an analytical fluorescence detection SEC-HPLC.
Identification of suitable detergent for membrane protein detergent extraction with the Wizard: TIME screen using FSEC. A) LDAO produces large, in-homogenous membrane protein particles, a sizeable fraction of which running in the ‘void’ volume. B) CYGLU-4 extracted membrane protein runs mainly as a symmetric single peak.
Here is a link to order info for the Wizard: TIME screen. More details on the FSEC protocols are described here:
Hattori M, Hibbs RE, & Gouaux E (2012). A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure (London, England : 1993), 20 (8), 1293-9 PMID: 22884106
KAWATE, T., & GOUAUX, E. (2006). Fluorescence-Detection Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins Structure, 14 (4), 673-681 DOI: 10.1016/j.str.2006.01.013
Final tip for dealing with detergent-solubilized membrane protein samples: no bubbles!