Protein biophysical measurement and amino acid analysis

Introduction

ITC is a thermodynamic technique that directly measures heat released or absorbed during biochemical binding events. It determines the relative binding affinity (Kd), reaction stoichiometry (n) and enthalpy change (ΔH). In addition entropy change (ΔS) and Gibbs free energy change (ΔG) and change in heat capacity of binding (ΔCp) can be determined. The data provides deeper insights into structure-function relationships and binding mechanisms.

More detailed information can be found from the manufacturer (Malvern) web page.

Sample requirements

• The sample and titrant must be in exactly the same buffer. Changing the sample buffer by dialysis or other suitable methods if there are differences in the buffer composition.
• Titrant concentration is usually 10x higher than sample concentration.
• Buffers with high ionization energy are not recommended. To obtain good results sample titration vs. buffer should be run as reference if there are any effects coming from the buffer.
• Required volume: ITC-200: titrant 50µl, sample 300µl

Prices

Academic service price: 50 EUR/hour

For frequent user, academic self-service, or company service prices please contact Dr. Hongmin Tu.

Introduction

Surface Plasmon Resonance (SPR) is a label-free technology for biomolecular interaction analysis. It monitors the formation and dissociation of biomolecular complexes on a sensor surface, where the ligand molecule is immobilized. The real-time binding measurement is performed by injecting the testing analyte in solution. This technology is widely used in ligand study, drug discovery and development. The facility is equipped with two different SPR instruments:

SPR - Biacore T200 (GE Healthcare)

Biacore T200 has measurement capacities ranging from 10e3-10e9/M/s for the association rate and 10e-1-10e-6/s for the dissociation rate. Minimum sample concentration is 10pM. This instrument also has a possibility to measure thermodynamic parameters.

MP-SPR – Navi-220A (Bionavis)

Navi-220A is a multiparametric SPR instrument that has extended capacities compared to the conventional SPR instruments. It is not limited only to the water based media in flow cell and bio-application. The application can expand to analyzinge substances in any media, e.g. water, gases and air. The chip surface could be modified in many different ways. Instead of the thin layer of gold in Biacore series Bionavis chips can be coated with any metals, organic or inorganic materials. This instrument ia able to provide more physical information about the surfaces as refractive indexes and layer thickness, etc. The measuring capacities range from 10e3-10e8/M/s for the association rate and 10e2-10e-5/s for the dissociation rate.

Sample requirements

All the SPR instruments are equipped with auto-sampler and racks for up to 96 samples. A standard analysis is composed of 4 steps:

1. Sample preparation – purification, dilutions etc.
2. Chip preparation – one of interactants must be immobilized on the chip surface using amine-, thiol-, aldehyde- coupling, or Tag-binding, e.g. His, Biotin, GST, etc. The ligand (immobilized on the chip) should have 100-200µl with a concentration of 1-100µg/ml. The immobilization buffer should not contain any “reactive groups”,
3. Injections – several injections of the analytes with different concentrations ranging from 0.1x to 10x of the expected binding affinity. Insert a chip regeneration step between each analyte injection. The running buffer for the interaction analysis can be phosphate, HEPES, or TRIS buffers. Minimum 100ml is needed for one experiment.
4. Data analysis – fitting with predefined model.

Click here for Biacore T200 system details.

Prices

Academic service price: Biacore T200, 100 EUR/hour; Navi 220A, 40 EUR/hour.

For frequent user, academic self-service, or company service prices please contact Dr. Hongmin Tu.

Uses of chips are not included in the price. The cost will be calculated separately according to the chip purchasing prices.

Introduction

Bio-Layer Interferometry (BLI) is another label-free technology that measures molecular interactions in real time for the purpose of detection, quantitation and kinetic analysis. Target molecules bind to the coated surface of the fiber-optic biosensors, forming a molecular layer that changes the spectral pattern of the reflected light. This technology is useful for basic research in protein-ligand interaction and for applications in biotherapeutic drug discovery and development. The facility is equipped with one advanced BLI instrument.

Octet Red 384 (ForteBio)

Octet Red 384 has measurement capacities ranging from 10e2-10e7/M/s for the association rate and 10e-6-10e-1/s for the dissociation rate. The affinity constant Kd ranges from 1mM to 10 pM. A number of specific biosensors are available from the manufacture.

Click here for Octet system details.

Sample requirements and sensor selection

Please discuss with the Core responsible person before the experiments.

Click here for more information.

Sample submission

Customers inside FBMM are suggested to use the in-house reservation system.
Customers from outside can contact the core responsible person by e-mail.

Prices

Academic service price: 55 EUR/hour.

For frequent user, academic self-service, or company service prices please contact Dr. Hongmin Tu.

Uses of sensors are not included in the price. The cost will be calculated separately according to the sensor purchasing prices.

Introduction

Circular dichroism (CD) spectroscopy is a form of light absorption spectroscopy that measures the difference in absorbance of right- and left-circularly polarized light by a substance. CD spectroscopy can be used to analyze the different secondary structural types: alpha helix, parallel and antiparallel beta sheet, turn, and others. The range is 1150 – 165nm.

We use a Chirascan™ CD Spectrometer (Applied Photophysics Ltd) for the measurements.

Click here for more information of the instrument.

Another type of analysis is thermal-denaturation. A series of CD spectra at different temperatures are recorded to study the melting point and thermodynamics of denaturation. Changes on the whole spectra, not only at certain wavelengths, are detected.

The instrument could also be used as a typical UV/VIS/nearIR spectrometer. We have an additional auto-titration unit for titration experiments.

In addition the instrument is equipped with a stopped-flow unit to measure reaction kinetics.

Sample requirements

• Typically 10mM phosphate buffer is used for far UV ranging up to 180-190nm. Buffers should not contain any groups which absorb in the far UV range.
• Samples should be in the same buffer as the reference for the baseline. Samples should not contain any “organic solvents” such as glycol, glycerin, DMSO, etc. Sample concentrations should be in a range of 5-100ug/ml. The concentration depends on the protein types. Typically 20-30ug/ml is enough to obtain good spectra. 250-300ul of sample volumes are needed for an 1mm-cuvette.

Click here for more detailed experimental protocols.

Prices

Academic service price: 40 EUR/hour.

For frequent user, academic self-service, or company service prices please contact Dr. Hongmin Tu.

Introduction

Amino acid analysis at Biocenter core facilities is composed of manual hydrolysis of protein or other biological samples, derivatization of the amino acids using Waters AccQ•Tag Chemistry, and HPLC analysis of the amino acid composition using Shimadzu Prominence LC system with a fluorescence detector.

Sample requirements

We require 1 to 5 μg of proteins or peptides for the analysis. The sample should not contain salts, detergents, and glycerol. We also accept samples blotted onto the PVDF membrane. If your protein or peptide amount is much lower than 1 μg, or your sample is not a purified protein or peptide, please discuss with us in advance.

Prices

Academic service price: 35 EUR/sample.

For frequent user, academic self-service, or company service prices please contact Dr. Hongmin Tu.

Introduction

NanoDSF is a label-free technique measuring the intrinsic fluorescence of a protein under either thermal or chemical denaturation processes. The technology is useful for study the protein stability.

The facility is equipped with one advanced NanoDSF instrument: Prometheus NT.48 (NanoTemper)

Click here for Prometheus NT.48 details.

Sample requirements and capillary selection

Please discuss with the Core responsible person before the experiments.

Sample submission

Customers inside FBMM are suggested to use the in-house reservation system.

Customers from outside can contact the core responsible person by e-mail.

Prices

Academic service price: 20 EUR/hour.

For frequent user, academic self-service, or company service prices please contact Dr. Hongmin Tu.

Uses of capillaries are not included in the price. The cost will be calculated separately according to the capillary purchasing prices.

Introduction

ThermoFluor is for measuring protein stability. The proteins are labeled with SYPRO Orange Fluorescent Dye. The facility is equipped with one CFX 96 Real-Time system (Bio-Rad) for this study.

Sample requirements

Please discuss with the Core responsible person before the experiments.

Sample submission

Customers inside FBMM are suggested to use the iLab reservation system. Customers from outside can contact the core responsible person by e-mail.

Prices

Academic service price: 20 EUR/hour. For frequent user, academic self-service, or company service prices please contact Dr. Hongmin Tu.