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BIOMOLECULAR CHARACTERIZATION FACILITY

Our mission is to provide access to biomolecular characterization services for Boise State investigators, external academic investigators, and industry partners. We can provide direct services, training and/or access to equipment for plasmid propagation, rapid protein solubility screen, circular dichroism and analytical ultracentrifugation. We are a fee-for-service center and use of our facility requires users to establish an ilabs account for reservations. To establish an account, or to inquire about our services and equipment, please contact:

Lisa Warner, PhD
Biomolecular Characterization Manager
Biomolecular Research Center
1910 University Drive
Boise, ID 83725-1511
Phone: (208) 426-2260
Email: lisawarner@boisestate.edu


SERVICES

Protein Expression and Purification
The first step in biomolecular characterization usually requires recombinant expression of proteins of interest. At the BRC, we provide services to propagate plasmids as well as a rapid, small scale screen for soluble protein expression in E. coli using up to 4 expression strains including basic BL21(DE3) and derivatives that can increase soluble expression of proteins with cell toxicity (e.g. enzymes), and with disulfide bonds, and from plasmids with rare codon usage.

Sample requirements

  • Plasmid propagation: Cell stock (e.g. plasmids from Addgene) or plasmid stock.
  • Small scale solubility screen: Expression plasmid with T7 inducible promoter.

Circular Dichroism
The BRC houses a Jasco J-810 Spectropolarimeter (163-900 nm). This hybrid instrument consists of a variable wavelength spectropolarimeter and absorption spectrophotometer. It is equipped with a computer-controlled Peltier device (3-90 °C) and a two-syringe titrator, which can be used to assess thermal/chemical stabilities of proteins/polypeptides or to monitor ligand binding.

CD applications include determining:

  • Optical purity of substance
  • Protein and nucleic acid secondary and tertiary structure
  • Conformational stability at varying temperature, pH, or denaturant concentrations
  • Conformational changes due to molecular interactions
  • Conformation of mutants or proteins expressed in different systems
  • Thermodynamics binding constants
  • Kinetics of folding and unfolding of macromolecules

 Buffer Requirements

  • Buffers that absorb in the spectral region of interest should be avoided.
  • 10 mM sodium or potassium phosphate buffer is generally recommended for CD. Tris or TEA can be used but should be pH’d with sulphuric or phosphoric acid.
  • Many buffers absorb at the shorter wavelengths where most of the structural information of interest is found. AVOID chloride, citrates, MOPS, imidazole and DTT.
  • Record a spectrum of the buffer alone before starting with samples to ensure the buffer absorbance is not a problem.

Sample Requirements:

  • The sample should be as pure as possible since impurities will contribute to the CD signal. Dialyzing into the buffer of choice is highly recommended.
  • The sample concentration is determined by the pathlength of the cuvette. The BRC has several choices.
    • A starting guideline is the sample should not exceed an absorbance of 0.9 OD over the wavelengths you want to scan. Measure the absorbance of your sample at all of the wavelengths you plan to use in your experiment to ensure your sample does not exceed 0.9 OD.
    • When using a 1 mm cuvette, 0.2 mg/ml for the 190-230nm range is a good starting estimation.
  • A smaller pathlength cuvette will decrease solvent absorbance to allow scanning down to lower wavelengths but will require a more concentrated sample.
  • Bring at least 500uL of sample and at least 10mL of identical buffer, ideally from dialysis.

Use and Management
The purpose of this instrument is to support research at Boise State University and surrounding research facilities. First-time users please contact the BRC for information on training and access requirements. Qualified users will be able to book instrument time and use the instrument independently.


Analytical Ultracentrifugation
Analytical ultracentrifugation relies on the simple premise that biomolecules in solution will sediment in the presence of the gravitational force applied by the centrifuge. The rate at which the particle sediments will be affected by its molecular weight, size and shape allowing one to determine these parameters experimentally. There are two basic types of experiments performed with the analytical ultracentrifuge, sedimentation velocity and sedimentation equilibrium.

  • Sedimentation velocity experiments rely on a high angular velocity which causes the solute to sediment rapidly leading to a depletion of solute near the meniscus. A boundary forms between the depleted and uniform concentration areas of the solute which can be monitored to determine the sedimentation coefficient which is a measure of the effective size of the solute.
  • Sedimentation equilibrium experiments employ a smaller angular velocity than sedimentation velocity which causes the solute to sediment at a much slower rate. As sedimentation occurs, diffusion opposes the gradual concentration increase in the bottom of the cell. After the two opposing forces reach equilibrium, the diffusional flow exactly balances the sedimentation flow leading to a concentration profile that is constant over time from which the molecular weight of the solute can be determined.

AUC applications include determining:

  • Sample purity
  • Molecular weight
  • Oligomer formation
  • Conformational changes
  • Ligand binding

Instrument Information:

  • XL-I (Beckman Coulter, Installed)
  • Rotors:
    • An-60 Ti – 4-place titanium rotor rated for 60,000 rpm
    • An-Ti 50 – 8-place titanium rotor rated for 50,500 rpm

Sample Requirements:
Sedimentation Velocity: At least 500uL of sample with an OD of 0.7-0.8 at the absorbance wavelength.
Sedimentation Equilibrium: At least 500uL of sample with an OD of 0.2-0.3 at the scanning wavelength.
**Bring at least 10mL of buffer as well.

Use and Management
The purpose of this instrument is to support research at Boise State University and surrounding research facilities. First-time users please contact the BRC for information on training and access requirements. Qualified users will be able to book instrument time and use the instrument independently.

For rate information please see BRC Recharge Rates found here.
Please contact Dr. Lisa Warner at 208-426-2260 or by email at lisawarner@boisestate.edu  for more information.