Biophysical Laboratory

About the Laboratory

The biophysical laboratory is a consortium of small instruments mainly used for characterization of biomolecules and biomolecular interactions. The instrumentation includes Circular Dichrosim, Dynamic Light Scattering, Isothermal Titration Calorimetry, and Surface Plasmon Resonance. This research lab has a very multi-disciplinary user base, and include researchers from colleges of Medicine, Agriculture and Bioresources, Engineering, Veterinary Medicine, and Physical Sciences, and Pharmacy & Nutrition.

Laboratory Contact

Location:
Room 192

Phone Number:
(306) 966-1712

Laboratory Managers:
Jason Maley

 

Biophysical Techniques in the Lab

Circular dichroism (CD) is a spectroscopic technique that measures the difference in the absorption (ΔA) of left-handed (AL) and right-handed (AR) polarized light which arise due to a molecule’s structural asymmetry. CD follows the Beer-Lambert Law: 

CD = ΔA = AL – AR = (εL-εR)bc

Where ε is the molar absorptivity of left-handed or right-handed polarized light, b is the optical pathlength, and c is the sample concentration. Although CD may be used for any molecule containing chiral chromophores, this technique has found widespread use for characterization of biomolecules (proteins, peptides, polynucleotides).

Typical applications of CD include:

Protein folding – evaluation of secondary structure, tertiary structure

Conformation stability – evaluation of proteins stability by thermal, pH, and chemical (urea, guanidine HCl) stability

Quality control in the laboratory – Does protein structure change from batch-to-batch? Does storage change conformation? Different solution effects (buffers, salts, detergents, etc.) on the conformation of biomolecules.

Kinetic – Stopped flow measurements for protein folding, denaturation, etc.

Protein-Protein Interactions

Instrument –Pistar-180 CD spectrometer 

The Applied Photophysics PiStar-180 spectrometer available at the SSSC is designed for both steady-state and kinetic CD applications. In addition to CD detection, the PiStar-180 spectrometer can also do measurements in absorbance, total fluorescence (use of suitable cut-off filters), temperature-controlled experiments, and stop-flow kinetics.

Light-Source:

75W Xe Lamp (75W HgXe Lamp available for kinetics)

Wavelength Range:

180 - 850 nm using MgF2 optics

Temperature Range:

5 - 95 °C (external circulator)

Kinetics:

Millisecond deadtime (measure rate constants up to 1500 s-1). 1:1 and 1:10 volume ratio injections are available

Dynamic Light Scattering (DLS), sometimes referred to as photon-correlation spectroscopy (PCS) or Quasi-Elastic Light Scattering (QELS), is a well-established technique that measures particle size distribution in the nanometre region. Particles in solution are constantly bombarded by solvent molecules, inducing the particle’s uncorrelated random motion, or Brownian motion. When the particle solution is illuminated by a laser, scattered light from the randomly moving particles adds both constructively and destructively leading to time-dependent intensity fluctuations. By using a fast photon counter, the time-dependent intensity fluctuations, which are directly related to the rate of diffusion of the particle through the solvent, can be measured. Thus, analyzing the time-dependent intensity fluctuations can determine the hydrodynamic radius of a particle using the Stokes-Einstein relation. 

Typical particles measured by DLS include:

  • Protein characterization, including aggregation, conformation, structural and thermal stability
  • Macromolecular complexes
  • Surfactant and micellular systems
  • Nanoparticles and colloidal dispersions
  • Biological and synthetic polymer characterization

Instrument – DynaPro MS800

The SSSC has a MS800 DLS instrument from Protein Solutions Inc. (now incorporated into Wyatt Technologies). This model is very sensitive, and is used for solutions containing particles with hydrodynamic radii (RH) in the 0.5 to 50 nm region. The DLS experiment is non-evasive, easy to use, and can provide size information of the particle in the order of minutes. The sensitivity of the instrument is quite high, where measurements are possible on as little as 100 mg mL-1 of a 14 kDa protein.

Instrument Specifications

Size Range

0.5 to 50 nm hydrodynamic radius

Laser Diode

830 nm (power adjustable)

Temperature Range

4 to 60 oC

Sensitivity

0.1 mg mL-1 for 14 kDa protein at 20 oC

Cuvette

Hellma 105.252-QS (b=1.5 mm, z=15mm, V=12 mL)

Isothermal titration calorimetry (ITC) is a biophysical technique that allows the thermodynamic study of two interacting species. When these two species interact, heat is either generated or absorbed. By measuring these interaction heats, binding constants (K), reaction stoichiometry (n), and thermodynamic parameters including enthalpy (DH) and entropy (DS) can be accurately determined. In addition, varying the temperature of the experiment allows the determination of the heat capacity (DCp) for the reaction. Thermodynamic data provides a molecular level insight into the forces that drive complex formation, including hydrogen bonding, hydrophobic interactions, charge-charge interactions, etc.

The basic principles of an ITC experiment are summarized in this Malvern short video. Some of the more common applications for ITC include biomolecular interactions (proteins, peptides, lipids, polysaccharides, small molecules, etc.). Other applications include surfactant characterization (cmc determination, etc.), enzyme kinetic assays, cell metabolism, nanoparticle interactions, soil particle interactions, and decomposition/stability of solid materials. 

Instrument – Calorimetry Sciences 4200 Isothermal Titration Calorimeter

The SSSC is equipped with a Calorimetry Sciences Corporation (now part of TA Instruments) ITC4200 microcalorimeter which is equipped with removable Hastelloy cell assemblies. The CSC ITC allows researchers to study almost any kind of interaction, including solutes with immobilized enzymes, tissue samples, or other solid materials in suspension.

Typical experiments take approximately 30 min to stabilize the baseline, and approximately 1-2 hr for data acquisition. There are standard binding models in the instrument’s software, or researchers can create more advanced models in Origin or other graphing software.

Instrument Specifications

Affinity Constant (Ka)

102 to 109 M-1

Competitive Binding Affinity Constant (Ka)

109 to 1012 M-1

Minimum Detectable Heat

0.1 mcal (0.4 mJ)

Baseline Stability

± 0.2 mcal s-1 hr (±0.08 mW hr-1)

Cell Volume

0.5-1.3 mL (full cells are recommended)

Precision Buret

25-250 mL

Volume Increment

1-20 mL (± 0.01 mL delivery precsion)

Stir Rate

0-500 rpm

Temperature Range

0-100 oC (Bath stability ±0.0005 oC at 25 oC)

Surface Plasmon Resonance (SPR) measurements offers research groups the opportunity of measuring biomolecular interactions in a real-time and label-free environment. SPR is an optical phenomenon which occurs at a metal/liquid interface and it is highly sensitive to changes in refractive index (measure refractive index differences on the order of 10-6!) near the surface, resulting in the ability to monitor mass changes (ie. binding events) occurring at the surface in real time. SPR experiments can characterize the following biomolecular interaction aspects: (1) specificity of interaction (yes/no); (2) rate of association (ka) (“recognition rate”); (3) dissociation rate (kd) (stability rate of complex); (4) affinity constant (KD) (ie. strength of interaction: Kd = kd/ka); (5) thermodynamics (SPR measurements completed at different temperatures). 

The common advantages of this technique include being a label-free technique, real-time biomolecular interactions, and the relatively small sample volume requirements. In addition, there are many different sensor chips available to immobilize a variety of tagged biomolecules (His-tags, biotinylated, etc.). However, the kinetics of an interaction can provide insight towards the biomolecular interaction.

Why are biomolecular interaction kinetics important? Isn’t a KD good enough?

The affinity constant, KD, is generally used to assess the strength of binding in a biochemical interaction. However, the kinetics of the interaction can reveal significant and meaningful information that cannot be obtained by other methods that evaluate only KD. Interactions with the same affinity can have different orders of magnitude association and dissociation rate constants (see Figure 1). For example, SPR kinetic measurements are used extensively in the pharmaceutical industry in order to evaluate potential drug candidates. An antibody or small molecule that may have a high affinity (low KD) but a high kd (fast dissociation) can be easily replaced in vivo, and would likely not a good drug candidate.

Figure 1. SPR sensorgrams of showing the changes in binding kinetics (ka, kd) profiles for different biomolecular interactions having the same affinity (Kd = 5.0 nM).using the same concentration of analyte (50 nM) and same affinity

Figure 1. SPR sensorgrams of showing the changes in binding kinetics (ka, kd) profiles for different biomolecular interactions having the same affinity (Kd = 5.0 nM).using the same concentration of analyte (50 nM) and same affinity

 

Instrument – Proteon XPR36

The Biorad Proteon XPR36 instrument located in the SSSC has a 6 x6 parallel flow channels (6 horizontal and 6 vertical flow channels) configuration. This flow design allows for fast experimental optimization, and high throughput screening (up to 36 different ligands immobilized on one sensor chip). The system is fully automated, so experiments can be programmed and instrument left unattended.

Instrument Specifications

Refractive Index Range

1.33 to 1.37

Number of Flow Channels

6 Horizontal, 6 Vertical

Number of Interaction Spots

36 (0.2025 mm2)

Number of Interspot References

42

Detection Temperature Range

15-40 oC (max. 10 oC below ambient temperature)

Baseline Noise

< 1 RU (< 20 kRu); < 1.5 RU (20-40 kRU)

Baseline Drift

< 1 RU/min (15-40 oC)

Flow Rate

25-200 µL/min

Sample Injection Volume

25-449 µL

Minimum Sample Volume

93 µL (35+25+8 uL for system+vial dead volume)

AutoSampler

2 96-well plates (8 x 12 configuration)

Buffer Valves

Switch between 2 different running buffers

Online Degasser

For Buffer system only

Detection Limits (Typical Values*)

Concentration

10-3 to 10-10 M

Association Rate Constant (ka)           

103 to 107 M-1 s-1

Dissociation Rate Constant (kd)     

10-5 to 10-1 s-1

Affinity (kinetic) (ka/kd)

104 to 1011 M-1

Affinity (steady state)

104 to 109 M-1

MW Limit*

201 Da

*Many experimental factors can expand or contract these working ranges and limits. Typical values achieved are listed.

Training Information

The CD spectrometer is open to any academic research group. SSSC personnel will first train researchers on the instrument and offer assistance in experimental development if needed. Training typically lasts a few hours and SSSC personnel will typically assist researchers with measuring/optimizing their first measurements. After training, researchers may use the instrument independently and reserve the instrument time through SSSC Evolution site. 

CD quality cuvettes (variable pathlengths) are available for general use, and small biophysical equipment available at the SSSC (pH meter, small centrifuge, etc.). Users are responsible for supplying their own reagents and consumables (pipette tips, Eppendorf tubes, etc.). Temporary storage of materials at the SSSC may be done with consent, but the SSSC is not responsible for any loss or damage to the materials stored at the SSSC.

The DLS instrument is open to any academic research group. SSSC personnel will first train researchers on the instrument and offer assistance in experimental development if needed. Training typically lasts 1-2 hr running a standard sample(s). Specific sample preparation and handling, as well as proper cuvette cleaning will be discussed to minimize potential artifacts. SSSC personnel will typically assist researchers with measuring/optimizing their first measurements. After training, researchers may use the instrument independently and reserve the instrument time through SSSC Evolution site.

The ITC instrument is open to any academic research group. SSSC personnel will first train researchers on the instrument and offer assistance in experimental development if needed. Training typically lasts about ½ day running a standard interaction, and the finer details on baseline stability and multiple sample handling will be discussed. SSSC personnel will typically assist researchers with measuring/optimizing their first measurements. After training, researchers may use the instrument independently and reserve the instrument time through SSSC Evolution site.

The SPR instrument is open to any academic research group. SSSC personnel will first train researchers on the instrument and offer assistance in experimental development if needed. Training typically lasts most of a day where we complete a standard SPR interaction (pH-scouting, ligand immobilization, analyte interaction). SSSC personnel will typically assist researchers with measuring/optimizing their first measurements. After training, researchers may use the instrument independently and reserve the instrument time through SSSC Evolution site.

Data Analysis Resources 

Common Consumables