Protein Purification and Molecular Interactions

This facility provides the platform for purification of proteins or biomolecules using FPLC systems. ÄKTATM pure M and ÄKTATMprime are available in the facility which will aid users in purification of proteins at preparative and analytical grades. This facility also provides the platform for measuring the biomolecular interactions using Biolayer interferometry and MicroScale Thermophoresis.

1. Bioreactor: New Brunswick BioFlo 415 SIP (7L & 14 L)

The BioFlo 415 is a versatile sterilization-in-place bioreactor that provides a fully equipped system in one compact package. It can be employed for batch or continuous culture with process control of pH, dissolved oxygen (DO), agitation, temperature, pump feed, anti-foam, level and addition analog/digital inputs and outputs.

2. Bio-Layer Interferometry (BLI): Octet RED96

Bio-Layer Interferometry (BLI) is a label-free technology for measuring biomolecular interactions. 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. 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. 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, Δλ. Unbound molecules, changes in the refractive index of the surrounding medium or changes in flow rate do not affect the interference pattern. This is a unique characteristic of BLI and extends its capability to perform in crude samples used in applications for protein:protein binding, quantitation, affinity and kinetics.

Assays can now be performed over a wider temperature range of 15-40°C on the Octet RED96 system, allowing for kinetics measurements of unstable proteins at lower temperatures or biologically relevant molecules at physiological temperatures. An evaporation cover for microplates also allows for greater walk-away run time, with minimal sample evaporation for up to 12 hours. The new Octet Data Analysis HT software version released with the Octet RED96 system brings custom reporting options and multi-experiment analysis that reduces analysis time from hours to minutes.

3. FPLC: ÄKTATM pure M

ÄKTA pure M is a flexible and intuitive chromatography system for fast purification of proteins, peptides and nucleic acids from microgram levels to tens of grams of target product. ÄKTA pure M is a reliable system where hardware and UNICORNTM software are designed to work together with columns and chromatography resins to meet purification challenges.

Depending on the column used with ÄKTA pure M, it can be used for the protein purification on the basis of charge (ion-exchange chromatography using columns: HiTrap Q, HiTrap SP etc.), affinity tags (affinity chromatography using columns: HisTrap, GSTrap, MBPTrap etc.) or size (size-exclusion chromatography using columns: HiLoad 16/600 Superdex 200 pg, etc.).

4. Multi Angle Light Scattering (MALS): Viscotek SEC-MALS 20

The Viscotek SEC-MALS 20 is a multi angle light scattering detector that can be used to measure the absolute molecular weight of molecules. It simultaneously measures the intensity of scattering at each angle which, in conjunction with a concentration detector, enables the calculation of the absolute molecular weight of proteins, synthetic & natural polymers and also molecular size expressed as the radius of gyration, Rg. For proteins, SEC-MALS measures the absolute molecular weight independent of column retention volume in order to study oligomeric state and aggregation.

5. FPLC: ÄKTATMprime

ÄKTAprime is the simple version of ÄKTA pure M. It is used to detect or purify biomolecules such as proteins in a mixture by recording the absorbance at 260 or 280 nm. The purified components of the mixture are collected in separate test tubes using a fraction collector. Ion-exchange chromatography, affinity chromatography, desalting, buffer exchange can be performed using this system.

6. MicroScale Thermophoresis (MST): Monolith NT.115

Monolith NT.115 uses MicroScale Thermophoresis (MST) technology to quantify binding events. One partner is typically labeled with a fluorescent dye and becomes an extremely sensitive reporter for all binding interactions. Monolith is all about accurately determining binding affinity in minutes. The strength of the interactions between a fluorescently labeled or intrinsically fluorescent sample and a binding partner (or ligand) are measured while a temperature gradient is applied over time. These events, weak or strong, cause a change in MST signal that is easily detected by plotting the normalized fluorescence against concentration of ligand. The results are automatically translated into precise and quantitative Kd values. Interactions can be studied in immobilization-free format.

7. Other services

The facility also provides services for the following instruments.

  • Sorvall Lynx 6000 centrifuge, ThermoScientific
  • FilterMax F5 Multi-Mode-Microplate-Reader, Molecular Devices
  • Sonics vibra cell sonicator
  • GEA Niro Soavi Panda Plus

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