Introduction

The IBC Biophysics facility maintains a variety of instruments for biophysical studies and provides consultation, training and instrument access for IBC investigators. The lab currently houses an isothermal titration calorimeter (VP-ITC, Microcal), an analytical ultracentrifugation (XL-A, Beckman), a CD (J815, Jasco), a Polarimeter (341, Perkin Elmer), a fluorescence spectroscope (Fluorolog-3, Jobin Yvon), a FT-IR (Tensor27, Bruker), a UV-VIS spectrophotometer (V630 Bio, Jasco), a Mulit-mode micro plate reader (M1000 pro, TECAN), a real-time PCR machine (Light Cycler 480II, Roche), a Muti-Angle Light Scattering (MALS) system (miniDAWN, Wyatt) and a Asymmetric Flow Field-Flow Fractionation (AF4) system (Dualtec, Wyatt). These equipments allow measuring the molecular weight, secondary structure contents and purity of biological macromolecules, as well as to study the affinity, stoichiometry, kinetic and thermodynamics between biomolecules and ligands.

The IBC Biophysical Instrumentation Laboratory is further integrated with the Biophysics Core Facility (BCF) of the Scientific Instrument Center, Academia Sinica, which is also hosted by IBC at the same location (Rm 402). For further information and access to the BCF instruments, please visit BCF homepage at http://bcf.daais.sinica.edu.tw/ or contact the manager in charge : Dr. Chris Jao .

01 恆溫滴定測焓儀 VP ITC 1

1. Isothermal Titration Calorimetry
Type:VP-ITC (Microcal)

Reservation (IBC)

Isothermal titration Calorimetry (ITC) is a powerful tool used to determine the thermodynamics of a particular reaction between two molecules. ITC directly measures the heat released or absorbed during molecular binding. In addition, there are no labeling or immobilization required and almost no buffer restrictions. Measurement of this heat enables direct accurate determination of a number of thermodynamic parameters in a single experiment: binding affinity (KD), stoichiometry (n), enthalpy (ΔH). From the former and the latter of these parameters we can derive as well the entropy (ΔS) of the reaction.

02 分析式超高速離心機 AUC 1 1

2. Analytical Ultracentrifuge, AUC
Type:XL-A (Beckman)

please contact manager

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.

03 定量即時聚合酶鏈鎖反應儀 Real time PCR 1

3.Real time PCR
Type:Light Cycler 480 II (Roche)

Reservation (IBC)

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.

04 傅立葉紅外線光譜儀 FT IR 1

4. FT-IR
Type:Tensor27 (Bruker)

Reservation (IBC)

Fourier Transform InfraRed(FT-IR) is a method of obtaining infrared spectra by first collecting an interferogram of a sample signal using an interferometer, and then performing a Fourier Transform (FT) on the interferogram to obtain the spectrum. An FT-IR Spectrometer collects and digitizes the interferogram, performs the FT function, and displays the spectrum. We have the TENSOR 27 FT-IR spectrometer which is fully digitized and provide high flexibility and sensitivity for liquid or solid samples.

05 螢光光譜儀 1

5. Fluorescence spectroscopy
Type:Fluorolog-3 (Jobin Yvon)

Reservation (IBC)

Fluorescence spectroscopy is a powerful tool for biological applications as it can be used quantitatively and is a more sensitive technique than absorbance. The FluoroLog-3 has a very high sensitivity and a unique modular system. With sensitivity and flexibility, the Fluorolog-3 can be applied in various biophysical studies such as FRET, molecular dynamics, or anisotropy decay experiments.

06 圓二色光譜儀 1

6. Circular Dichroism spectroscopy
Type:J-715 (Jasco) – The verticalized J-715 is more suitable for precipitating samples.

Reservation (IBC)

Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light of chiral molecules. CD is very sensitive to the secondary structure of polypeptides and proteins, so characterization of the protein secondary structure is the most popular application. CD spectroscopy can be also used to determine protein folding, detecting structure changes in structure upon mutagenesis and studying pH、temperature and denaturant stability of biomolecules. Notably, our J-715 has been verticalized and it is suitable for precipitating samples.

07 旋光光度計 1

7. Polarimeter
Type:341 (Perkin Elmer)

Reservation (IBC)

Chemical substances are usually optically active, and polarized light will rotate either to the left or right when passed through these substances. The amount by which the light is rotated is known as the angle of rotation. Polarimeter is a scientific instrument used to measure the angle of rotation caused by passing polarized light through an optically active substance. It is a highly versatile instrument for research applications as well as for quality control work.

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8. Glycostation Reader
Type:Glycostation Reader 1200 (GP Biosciences)

Reservation (IBC)

Glycans play a significant biological role in biomedical areas; cancer, immunological disease, infectious disease and glycoproteins show promise in the area of cancer biomarkers. Today, approximately 50% of current cancer markers are various types of Glyco-markers. Glycostation Reader 1200 is a novel glycan profiling instrument based on the principle of evanescent-field fluorescence excitation in combination with lectin microarrays. It provides a quick, simple and highly sensitive method for profiling and analyzing complex glycan structures of biomarkers, glycoproteins and stem cell.

10 圓二色光譜儀 1

9. Circular Dichroism spectroscopy
Type:J-815(Jasco)- It equipped with the single position Peltier temperature control system and the auto-titration system and the Secondary Structure Estimation (SSE) program

Reservation (IBC)

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.

11 停流反應光譜儀 1

10. Stopped-Flow Spectroscopy
Type:SX18 (Applied Photophysics)

Reservation (IBC)

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.

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11. Tecan Infinite M1000 pro
Type:Infinite M1000 pro (Tecan)

Reservation (IBC)

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.

13 粒徑篩層析 多角度散射分析系統 1

12. Size Exclusion Chromatography- Multi-Angle static Light Scattering (SEC-MALS)

please contact manager

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)
14 紫外光 可見光分光光度計 1

13. UV-Vis Spectrophotometer
Type:V-630-Bio (Jasco)

Reservation (IBC)

The V-630Bio is a specifically designed instrument dedicated to Life Science applications. An intelligent Remote Module (iRM) designed for biological and clinical analysis, and a micro cell holder. The iRM conveniently guides the operator through routines encompassing data acquisition to data processing. Built-in bio-analytical application programs include: protein/nucleic acid measurement; temperature ramping/DNA melting analysis; kinetics measurement/analysis; and a quantitative protein are installed and ready for analysis.

Specifications:

  • Double-beam spectrophotometer with single monochromator
  • Silicon photodiode detectors
  • Range 190 to 1100 nm
  • Fixed bandpass of 1.5 nm
  • High-speed scanning up to 8,000 nm/min
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14. Asymmetric Flow Field-Flow Fractionation
Type:Eclipse DualTec (Wyatt)

please contact manager

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.

16 全功能盤式判讀儀 1

15. BMG CLARIOstar
Type:CLARIOstar (BMG)

Reservation (IBC)

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 HORIBA 1

16. Duetta
Type:Duetta (HORIBA)

Reservation (IBC)

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.

A01 盤式動態光散射儀 1

A01. DynaPro Plate Reader I
Type:DynaPro Plate Reader I (WYATT)

please contact Dr. Meng-Chiao Ho's Lab

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.

A02 定量即時聚合酶鏈鎖反應儀與蛋白質熱穩定分析軟體 1

A02. StepOnePlus Real-Time PCR System
Type:StepOnePlus Real-Time PCR System (Applied Biosystem)

please contact Dr. Meng-Chiao Ho's Lab

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.

A03 個人化生物分子交互作用分析系統 1

A03. BLItz
Type:BLItz (Forte-Bio)

please contact Dr. Meng-Chiao Ho's Lab

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.