Magic™ Membrane Protein Functional Validation

Empowered by our cutting-edge analytical platforms and experienced technical personnel, Creative Biolabs is fully competent and dedicated to serving as your one-stop-shop for functional validation of membrane proteins.

Membrane proteins, such as G-protein coupled receptors (GPCRs) and ion channels, are the top one popular drug targets in therapeutic fields. However, the production procedure of membrane proteins is tedious and their structures are intrinsically complicated, which could potentially lead to loss of native activity. Therefore, prior to further application, information regarding basic functional activity is of great essence for proper evaluation of the membrane protein quality and the proceedings and directions of the project. Creative Biolabs offers a comprehensive set of functional analysis services to assist our clients to determine the in vitro characteristics of various membrane protein.

We provide versatile technologies to offer customers flexible choices:

• Ligand binding assay -- (labeled ligand binding assay & label-free ligand binding assay)
• Radioactive ligand-binding assay (RBA): As the current standard method for the validation of ligand interaction with GPCRs, it is relatively simple but powerful. It can provide sensitive and quantitative information about GPCR affinity for a wide variety of ligands. Three basic types of radioligand-binding experiments are available: saturation, inhibition and kinetic assays.
• Fluorescence-based assay: With wide spectrum of wavelengths, multiple colors can be applied for detection of a specific target. With more stable probes and more sensitive instruments, we can perform various experiments such as fluorescent intensity measurement, fluorescence polarization and energy transfer between probes to evaluate ligand binding properties.


Fig 2 Different binding assay methods. (Martin C, et al. 2011)

• Enzyme-linked immunosorbent assay (ELISA): Sandwich ELISA represents a most classic method for ligand binding profiling. It is label-free, easy-to-operate and compatible with high throughput format.
• SPR/BLI: Both surface plasmon resonance (SPR) and bio-layer interferometry (BLI) are label-free and capable of measuring real-time quantitative binding affinities and kinetics for membrane proteins interacting with ligands.


Fig 3 The principle of SPR (left panel) and BLI (right panel) technologies.

• Tag-lite assay: Tag-lite ligand-binding assay is non-radioactive and widely applicable to a whole range of multi-pass membrane proteins. This strategy is based on the combination of a homogeneous time-resolved fluorescence detection method with a covalent labeling technology called SNAP-tag, CLIP-tag, and Halo-tag. Tag-lite also offers adequate freedom and flexibility to build up your assays from target construction to assay development.


Fig 4 Principle of tag-lite technology.

• Isothermal titration calorimetry (ITC): Due to its high sensitivity and label-free property, ITC represents the gold standard of understanding the thermodynamics of biomolecular binding processes. It helps to evaluate the binding enthalpy variation by sensing heat alternations caused by the binding reaction. Moreover, ITC can be used to distinguish ligands with similar binding affinities by detecting differences in binding modes. In particular, this method is very reliable for entropy-driven ligands. It has been broadly used to investigate functions of GPCRs and ion channels in detergent micelles.


• Electrophysiology -- particularly suited for the investigation of transporter and ion channel function.
• Patch clamp: This label-free, highly sensitive method is generally considered as the ‘gold standard’ for assessing ion channel functions. It can measure biophysical and pharmacological properties of voltage-gated and ligand-gated ion channels present in cells, membrane patch or tissues with unmatched temporal resolution.
• Electrophysiology using planar lipid membranes: By absorbing proteoliposomes/membrane fragments onto solid supported membrane, we can analyze functional activities of many transporter proteins. This approach is much simpler and more efficient compared with the incorporation of the protein into the planar membrane, leading to a superior signal-to-noise ratio and time resolution of the electrical measurement.



• Membrane potential sensitive dye: There are many fluorescent dyes that are capable of translating membrane potential changes to fluorescent signal. Integrated with many voltage-sensitive dyes with a wide timescale ranging from microseconds to seconds, we can provide in-depth investigations to meet different needs. It can also help to measure over-expressed membrane protein functions in living whole cells in a noninvasive way.


• CPM assay: CPM is a thiol-reactive coumarin and short for 7-Diethylamino-3-(4'-Maleimidylphenyl)-4-Methylcoumarin. It is barely fluorescent unless specifically reacted with thiols, forming a conjugate with strong fluorescence. Therefore, any fluorescence signal emitted from the sample is specific to membrane proteins since neither scaffold proteins nor phospholipids have cysteines. This simple and high throughput assay is very compatible for studying the functionality/stability of membrane proteins in nanodiscs.

The information gathered from the initial step of analysis provides a guideline for membrane protein quality assurance and the downstream applications. Aided by this versatile platform, scientists of Creative Biolabs are glad to tailor specific functional validation service packages to fit your timeline and R&D budget. Please contact us for more information and a detailed quote.

Reference

1. Martin C, Orestis F, Jurriaan M Z, et al. (2011). Original Fluorescent Ligand-Based Assays Open New Perspectives in G-Protein Coupled Receptor Drug Screening. Pharmaceuticals (Basel). 4(1), 202-214.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.