Institute of Biological Chemistry, Academia Sinica

The analytical ultracentrifuge (AUC) contains an optical system allowing one to observe the movement of a sample and determine its sedimentation coefficient. As a versatile and powerful tool, AUC has been useful in characterizing the solution-state behavior of proteins and other macromolecules. The primary uses of AUC are: examination of sample purity、molecular weight determination、analysis of associating systems and detection of conformational changes. 
The AUC(Beckman Coulter, Model XL-A) is located in IBC room402 and shares 50% of the instrumental time with The Biophysics Core Facility (BCF) of the Scientific Instrument Center, Academia Sinica. Please contact the manager for further information.

We have the Roche LightCycler 480 II (LC 480 II) machine for quantitative PCR analysis. The LC 480 II is a high throughput gene quantification or genotyping real-time PCR platform with exchangeable blocks for 96 and 384 samples in multiwell plates. The LC 480 II uses a Xenon lamp as its light source and has a number of possible excitation/emission wavelengths to choose from, making multiplexing much easier. Thermo stability test of proteins is also available on this machine.

Circular dichroism (CD) spectroscopy is widely used to study chiral molecules of all types and sizes. The primary use is in analyzing the secondary structure or conformation of macromolecules, particularly proteins as secondary structure is sensitive to its environment, temperature or pH. CD spectroscopy can be used to observe how secondary structure changes with environmental conditions or on interaction with other molecules. Our J-815 CD spectroscopy is equipped with the single position Peltier temperature control system and the auto-titration system for various experiments. Furthermore, the Secondary Structure Estimation (SSE) program is installed and the helix, sheet and random coil contents of unknown proteins can be quantitatively estimated from CD spectra.

Stopped-flow is a spectroscopic technique used for studying fast reaction mechanisms in solution over timescales of about 1ms up to 100’s seconds. In general, two reagents are rapidly mixed together and then ‘stopped’ in an observation cell. The sample cell is irradiated with monochromatic light and as the reaction precedes the change in the recorded signal, usually a fluorescence signal or the absorbance at a specific wavelength, is recorded as a function of time. Analysis of the resulting kinetic transient can determine reaction rates, complexity of the reaction mechanism, information on short-lived reaction intermediates etc. A series of stopped-flow experiments can be used to show the effect of parameters such as temperature, pH and reagent concentration on the kinetics of the reaction.

The Tecan Infinite M1000 pro is a multifunctional monochromator based microplate reader that provides high performance for the vast majority of today’s microplate applications and research. The Infinite M1000 pro in IBC is fully equipped and provides users with the following techniques: Absorbance, Fluorescence (top and bottom), Time Resolved Fluorescence, Fluorescence polarization, Luminescence (Glow type, Flash type and Dual-color), Luminescence scan and an external injector module for further applications.

The Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS) is an established analytical method for the absolute determination of molar mass of large molecules. The system is composed by a HPLC (Agilent) coupled with four “on-line” detectors: laser light scattering (miniDAWN TREOS, Wyatt), Quasi-elastic light scattering (QELS, Wyatt), refraction index (Optilab T-rEX, Wyatt) and ultraviolet detection. MALS combines the precise measurements of light scattering intensity and concentrations for a rigorous calculation of solution-property molecular weights—making no assumptions of conformation or shape, and no perturbations to the sample. In addition, QELS module can further provide simultaneous hydrodynamic radius (Rh) measurements in the same scattering volume viewed by the MALS detectors. The combined SEC-MALS method has many applications, such as the confirmation of the oligomeric state of a protein, quantification of protein aggregation, and determination of protein conjugate stoichiometry.

Type：

• Static/Dynamic Light Scattering: miniDAWN TREOS with QELS (Wyatt)
• Refractive index detector: Optilab T-rEX (Wyatt)
• HPLC: 1260 Infinity Quaternary LC System (Agilent)

Asymmetric Flow Field-Flow Fractionation (AF4) is a size-based technique to separate larger molecules (i.e. proteins oligomers, polysaccharides, liposomes and nanoparticles) in the size range of 1 to 1000nm. Because the separation occurs without shear or adverse, non-ideal column interactions, AF4 complements size exclusion chromatography (SEC) for macromolecular separations. In addition, the AF4 conjugated our available set of detectors can be applied to characterize the molar mass and hydrodynamic radius of soluble particles.

CLARIOstar is ahigh-performance multi-mode microplate reader and equipped with LVF Monochromators, spectrometer, and filters. LVF Monochromator provides the highest flexibility while delivering filter-like performance for fluorescence. In addition, the CLARIOstar incorporates an ultra-fast spectrometer for UV / Vis absorbance detection. This technology allowed users to capture a full spectrum from 220 to 1000 nm in less than 1 second per well and most absorbance assays will benefit from full spectrum analysis.

Duetta is a spectrofluorometer that combines fluorescence and absorbance spectrometers. Duetta uses the high-speed built-in CCD detector that can acquire a full fluorescence spectrum from 250 nm to 1,100 nm in less than one second. The Duetta in IBC also equipped a self-identifying four position Peltier holder (1 cm) for automated temperature control and ramping from -15 ˚C to +110 ˚C, with magnetic micro-stirrers under all four positions.

The Thermo Scientific Nicolet iS50 FTIR Spectrometer is a versatile instrument designed to meet the demands of advanced analytical and research applications. Known for its innovative design and exceptional performance, the iS50 simplifies complex spectroscopy tasks, making it the ideal solution for materials analysis, quality control, and academic research. Equipped with a fully integrated and upgradeable design, its modular approach allows users to customize the system with advanced options. The Nicolet iS50 offers multiple sampling capabilities, including transmission, reflection, and ATR (attenuated total reflectance). 

Specifications:

• Spectral range: 7800~350 cm^-1
• Signal to noise ratio: Better than 55,000 : 1
• Spectral resolution: Better than 0.09 cm-1 
• Beam splitter: Ge-on-KBr
• Detector: DTGS and MCT detectors are installed.
• Source: Polaris long-lifetime mid-IR source

The DynaPro Plate Reader provides superior high throughput dynamic light scattering performance. It measures samples directly from industry-standard microplates (96, 384, or 1536 well plates) using as little as 5 microliters of sample per well. Once the data are collected the samples can be recovered and the plates discarded, no cleaning required. In addition, DynaPro has the temperature control peltier that can perform accelerated stability, thermal melting, and temperature studies of multiple samples in parallel.

The StepOnePlus Real-Time PCR System with the Protein Thermal Shift Software is suitable for high-throughput protein thermal stability analysis. The protein thermal shift applications include: developing protein formulations and storage buffers (pH, buffer and salt), analyzing effects of mutations or ligand-protein interactions and performing protein preparation quality control.

BLItz is an assay system for measuring biomolecular interactions by using BLI (bio-layer interferometry) technology. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. The binding between a ligand immobilized on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip, which results in a wavelength shift, which is a direct measure of the change in thickness of the biological layer. Interactions are measured in real time, providing the ability to monitor binding specificity, rates of association and dissociation, or concentration, with precision. The system only requires 4 μl of a sample and any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time.