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Membrane Proteins Affinity Measurement

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Membrane proteins play significant role in many biological processes including ions transportation, energy transduction, signal recognition and catalysis of chemical reaction. However, due to the difficulties in obtaining high-resolution protein structures in natural status, it is challenging to detect ligand-receptor interaction of membrane proteins. Based on our classical SPR platform, Creative Biolabs can carry out the membrane protein interaction assay in a native or native-like environment and high throughput manner.

Surface plasmon resonance (SPR) is one of the most powerful techniques for the study of molecular interactions. It is capable of measuring real-time binding affinity and kinetic in a label-free mode, which requires the ligand to be immobilized on a sensor chip, while allows the soluble analyte to flow across the sensor surface in the running buffer. The process is monitored by an optical element that can detect the tiny changes in refractive index near the surface. Normally, membrane proteins are difficult to be immobilized onto the sensor surface without affecting its activity. Thus, a lipid-reconstituted or detergent-solubilized conditions that can retain the membrane proteins in its active conformation are always required. Depending on the specific characteristics of each membrane protein, we provide certain different ligands to form the first layer on the chip in order to capture the membrane proteins at an appropriate orientation. With the receptor molecules attached to the first layer, ligands at a known site distinct from the interesting site help to orient the membrane protein towards the analyte flow rather than towards the sensor surface. To evaluate the specific binding interactions, positive and negative controls will be used before the formal measurement.

Figure 1. Immobilized rhodopsin for monitoring G protein activation.Figure 1. Immobilized rhodopsin for monitoring G protein activation.

Many candidates that bind to membrane proteins are small molecules (molecular weight < 1000 Da). Since the SPR effect depends on the detection of a mass change at the sensor surface, it always be a challenge thing to use a relative small-molecule as the free ligand. However, our scientists have extensive experience in balancing the ligand density for maximum activity and minimum denaturation.

Though expressing membrane protein in bilayer is preferable for affinity analysis, in some cases, to use a sample dissolved in detergent might be sufficient for an approximate measurement. To choose a cost-effective procedure, we are able to perform a Biacore-based assay to figure out the membrane proteins solubility. However, be different from cell-based assay, this test has to be evaluated case by case, as certain molecules, for example CCR5 and CXCR4, show similar KD values in the existence of detergent.

Creative Biolabs provides state of the art surface plasmon resonance microscopy (SPRM) that enables the measurement of binding kinetics of membrane proteins within a single living cell which keeps their original membrane environment rather than the artificial lipid bilayer or detergent solutions. Briefly, cells are directly cultured on the chip and the image is collected via an inverted microscope, which significantly enhances the sensitivity compared to traditional SPR.

Figure 2. Next-generation SPR instrumentation for measuring membrane protein-ligand binding.Figure 2. Next-generation SPR instrumentation for measuring membrane protein-ligand binding.

Reference:
Patching S G. Surface plasmon resonance spectroscopy for characterisation of membrane protein–ligand interactions and its potential for drug discovery[J]. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2014, 1838(1): 43-55.




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