Instrument: Octet RED96 (Sartorius)

Biolayer interferometry (BLI) is a label-free technology used to study biomolecular interactions in real-time. It operates on the principle of interferometry, where changes in the optical thickness of a biomolecular layer at the sensor surface result in interference patterns. BLI involves the immobilization of one binding partner (ligand) onto a biosensor tip. As the tip interacts with the other binding partner (analyte), changes in interference patterns occur, allowing for the determination of binding kinetics, affinity, and specificity. This technique offers advantages such as rapid data acquisition, minimal sample consumption, and versatility in studying various biomolecular interactions.

Instrument: J-815 CD Spectrophotometer (JASCO)

Circular dichroism (CD) spectroscopy is a powerful technique used to analyze the structural characteristics of chiral molecules, particularly proteins and nucleic acids. It relies on the differential absorption of left and right circularly polarized light by chiral molecules. When circularly polarized light passes through a sample containing chiral molecules, the absorption of one polarization state is greater than the other due to the molecule's inherent chirality. By measuring the difference in absorbance between left and right circularly polarized light as a function of wavelength, CD spectroscopy provides valuable information about the secondary, tertiary, and quaternary structures of biomolecules. This technique is widely employed in structural biology, biochemistry, and pharmaceutical research to study protein folding, ligand binding, and conformational changes.

Instrument: CFX96 qPCR (Bio-Rad)

Differential scanning fluorimetry (DSF) or Thermal Shift Assay uses a fluorescent dye to measure protein conformational changes and unfolding by tracking changes in fluorescence as a function of temperature. The fluorescent dye binds to hydrophobic regions on the protein as it begins to unfold with increasing temperature. Many different dyes can be used, but the most common dye to start with is SYPRO Orange. DSF is commonly used to study protein stability across many buffer conditions and to discover small molecule ligands in drug discovery. Our facility stocks the DSF screens listed below:

• Solubility and Stability Screen (Hampton)
• Solubility and Stability 2 Screen (Hampton)
• RUBIC Buffer Screen (Molecular Dimensions)
• Durham pH Screen (Molecular Dimensions)
• Durham Salt Screen (Molecular Dimensions)

Instrument: DynaPro NanoStar (Wyatt)

Dynamic light scattering (DLS) is a useful technique to measure the size distribution of biomolecules in solution. Sample homogeneity can be rapidly assessed and the hydrodynamic radius determined. DLS measurements are size and shape dependent, so molecular weight estimates may not be accurate. More accurate measurements of molecular weight can be made from static light scattering (SLS). Light scattering is routinely used to assess both protein aggregation and stability.

Instrument: TwoMP (Refeyn)

Mass photometry (MP) is a single-molecule technique that enables the direct measurement of mass and size distributions of unlabeled biomolecules in solution. It works by exposing molecules to a beam of light, and as the single molecules make contact with a glass surface they scatter some of the light. Light that isn't scattered is reflected by the surface. The interference between the light scattered by a molecule and the light reflected by the measurement surface is measured and is directly proportional to the molecule’s mass. The technique requires minimal sample and can quickly assess sample heterogeneity and quality.

Instrument: C-Trap (Lumicks)

The C-Trap single-molecule microscope combines optical tweezers, fluorescence, label-free microscopy, and microfluidics in a nicely integrated solution. Optical tweezers use a highly focused beam of light to trap beads, which can be coated with various biomolecules. Tiny forces can be applied to these molecules and then measured and/or visualized to understand the impact on interactions. Find more info and applications here.

Size-Exclusion Chromatography (SEC) separates macromolecules in solution by size and shape. The macromolecular separation profile is determined by various bead pore sizes for the specific column. Large particles will pass through the column more quickly and elute before smaller molecules, which diffuse into the pores and therefore elute later in the run. For globular proteins, SEC can be used to estimate the mass and oligomeric state of the sample by comparison to a set of MW standards.

Columns: 

• Superdex 75 (24 and 120 mL)
• Superdex 200 (24 and 120 mL)
• Superose 6 (24 and 120 mL)

Instrument: HT7800 (Hitachi)

We routinely perform and train users in negative stain EM using the microscope available in the Central Microscopy Research Facility (CMRF) at Iowa. Negative stain EM is a rapid technique to assess particle quality and homogeniety prior to cryo-EM.

The PCF houses a Vitrobot Mark IV and a custom Back-it-up device for grid preparation. Microscope time and sample shipments for grid screening and data collection is organized by the PCF at external facilities. Screening and data collection at the National Centers for Cryo-EM can also be facilitated through the PCF. Contact the Facility Director to inquire about potential projects.

The University of Iowa belongs to the Molecular Biology Consortium which operates beamline 4.2.2 at the Advanced Light Source (ALS) synchrotron within Lawrence Berkeley National Lab. Considerable beamtime is available through our consortium, so all of our crystal screening and data collection is performed remotely at ALS. Contact the Facility Director for questions about access.

ALS beam status

ALS operating schedule

Small-angle x-ray scattering (SAXS) experiments are performed on the BioCAT beamline at the Advanced Photon Source (APS) synchrotron at Argonne National Lab. We routinely perform SEC-SAXS and SEC-MALS-SAXS, but all available configurations can be found here. User proposals must be submitted to obtain beamtime.