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Energetic analysis of membrane protein folding and stability by Differential scanning calorimetry (DSC) (CAT#: STEM-MB-0523-WXH)

Introduction

The familial origin of membrane proteins is suficiently broad and encompasses a variety of sources including bacterial, viral, eukaryotic plasma or organelle membranes, as well as specific soluble proteins that undergo membrane insertion during a deWned stage within the cell cycle. Parallel structural and functional studies have revealed that membrane protein families are integrally involved in a multiplicity of specialized roles spanning the range from enzyme activity, cell signaling, receptors, and channels. Membrane proteins therefore assume a pivotal role in terms of regulating speciWc functions within the organelle, cell, or viral particle. In addition to the extraordinary number of membrane proteins that regulate important functions within the mammalian cell, both bacterial and viral pathogens have developed sophisticated strategies to manipulate the host cell life cycle to their own advantage by utilizing highly specialized.




Principle

Calorimetry is a primary technique for measuring the thermal properties of materials to establish a connection between temperature and specific physical properties of substances and is the only method for direct determination of the enthalpy associated with the process of interest. Calorimeters are used frequently in chemistry, biochemistry, cell biology, biotechnology, pharmacology, and recently, in nanoscience to measure thermodynamic properties of the biomolecules and nano-sized materials.
Amongst various types of calorimeters, differential scanning calorimeter (DSC) is a popular one. DSC is a thermal analysis apparatus measuring how physical properties of a sample change, along with temperature against time.1In other words, the device is a thermal analysis instrument that determines the temperature and heat flow associated with material transitions as a function of time and temperature. During a change in temperature, DSC measures a heat quantity, which is radiated or absorbed excessively by the sample on the basis of a temperature difference between the sample and the reference material.

Applications

Energetic analysis of membrane protein folding and stability.

Procedure

1. Instrument Start-up
2. Sample Preparation
(1) Dialyze the sample against the buffer that will be used as the reference for the experiment.
(2) Determine the concentration of the protein sample using the most suitable protein concentration determination method.
(3) Degas the sample and reference buffer in vacuum to get rid of microbubbles that can cause volume inaccuracy.
(4) load the samples and their respective buffer in pairs into 96 well plates compatible with the instrument.
(5) Place the plate in the sample holding compartment in the proper orientation.
3. Experimental Parameter Setup
Set the starting temperature, final temperature and the scan rate of the experiment.
4. Data Analysis

Materials

Differential Scanning Calorimeters