Instrumentation


Circular Dichroism (CD) Spectroscopy

Aviv Model 420SF CD Spectrometer

Instrument: J-1500 CD Spectrophotometer (Jasco)

Applications:

  • Conformational analysis of biomolecules
  • Protein thermal stability
  • Binding studies

Major Specifications:

  • Range: 163-950 nm
  • Light source: Xe 150W, supracil, air-cooled
  • Wavelength accuracy: 0.1 nm at 163-250 nm
  • Scan modes: continuous scan, step-scan, auto-scan
  • RMS noice: less than 0.004 mdeg at185 nm
  • Temperature regulation: –30 to +130° C

Probe:

  • Concentration: ~ 0.1-1 mg/mL
  • Cuvette volume: 2-4000 µL
  • Path length: 0.001-10 mm

Time for 1 analysis (spectrum): as low as 0.33min.

Fluorescence Spectroscopy

Single Photon Counting Spectrofluorometer FluoroMax-4C (HORIBA Instruments)

Instrument: Single Photon Counting Spectrofluorometer FluoroMax-4C (HORIBA Instruments)

Applications:

  • Macromolecular binding
  • Conformation data (based on environmental sensitivity, polarization, energy transfer, quenching studies, etc.)
  • Protein folding

Major Specifications:

  • Range: 200-950 nm (excitation and emission)
  • Light source: 150-watt Xenon arc lamp
  • Wavelength accuracy: 0.5 nm
  • Scan speed: up to 200 nm/sec
  • Water Raman signal-to-noise ratio: 3000:1
  • Temperature regulation: Peltier-based, -40 to +110°C
  • Integration time: 1 ms - 160 s
  • Sensitivity dynamic range: 6 orders of magnitude in the same spectrum
  • Polarization: automated
  • Well plate reader: from 6 to 384 samples at a time

Probe:

  • Cuvette volume: 30-4000 µL
  • Path length: 1-10 mm
  • Concentration: < 1 µM

Time for 1 analysis (spectrum): 1-5 min.

TCSPC (Time-Correlated Single Photon counting) unit: lifetime range from 25 ps to 10 s.

Isothermal Titration Calorimetry

iTC200 Isothermal Titration Calorimeter (MicroCal)

Instrument: iTC200 Isothermal Titration Calorimeter (MicroCal)

Applications:

  • Complete characterization of the molecular interactions of small molecules, proteins, antibodies, nucleic acids, lipids and other biomolecules in a single titration experiment
  • Enzyme kinetics
  • Assessment of the effect of molecular structure changes on binding mechanisms

Major Specifications:

  • Direct measurement of sub-millimolar to nanomolar binding constants
  • Temperature range: 2 to 80°C
  • Cell/cell compensation: power feedback
  • Response time: 10 seconds

Probe:

  • Cell: at least 2 µM of biomacromolecule in 200 µL of buffer solution
  • Syringe: 10 times more concentrated ligand in 40 µL of the same buffer

Time for 1 analysis (spectrum): as low as 0.5 hour, typically – 1 hour

LC-MS-MS High-Resolution Tandem Mass Spectrometry (Q-TOF)

6520 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS/MS (Agilent Technologies, Inc.)

Instrument: 6520 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS/MS (Agilent Technologies, Inc.)

Applications:

  • Identification, characterization, structure confirmation and profiling of samples as diverse as small molecules, metabolites, peptides, proteins, oligonucleotides, etc.

Major Specifications:

  • Typical mass accuracy: 1ppm MS and 2ppm MS/MS
  • Enhanced mass resolution: up to 20,000 resolving power
  • Ion source modes: Dual-spray electrospray ionization (ESI) source
  • Multimode source: ESI/APCI (atmospheric pressure chemical ionization)
  • In-spectrum dynamic range: up to 5 orders
  • Sensitivity: high-attomole to low-femtomole
  • Spectral acquisition rates: up to 20 MS or 10 MS/MS spectra per second
  • Time-of-flight mass range: m/z 20–20,000

HPLC-Electrospray Mass Spectrometry (Single Quad)

6100 Series Single Quadrupole LC/MS (Agilent Technologies, Inc.)

Instrument: 6100 Series Single Quadrupole LC/MS (Agilent Technologies, Inc.)

Applications:

  • Molecular weight and structure characterization

Major Specifications:

  • Mass range: m/z 10-3000
  • Mode: API-electrospray or Multimode (ESI/APCI)F
  • Mass accuracy: 0.13 Da within the calibrated mass range in scan mode
  • Mass axis stability: 0.13 Da over 8 hours
  • Sensitivity: for 1 pg reserpine RMS 100:1
  • Analyte polarity: from intermediate non-polar to highly polar
  • Multiple signal acquisition: ability to cycle through four different acquisition modes

Time for 1 analysis (spectrum): 5-40 min

Dynamic Light Scattering (DLS)

DynaPro NanoStar Dynamic Light Scattering instrument (Wyatt Technologies)

Instrument: DynaPro NanoStar Dynamic Light Scattering instrument (Wyatt Technologies)

Applications:

  • Characterization of protein size and size distribution, weight-average molecular weight and thermal stability of biomacromolecules
  • Screening samples for self-association over a broad range of solutions and conditions
  • Measurement of stability and size of lipids, conjugates, nanoparticles and other drug delivery particles

Major Specifications:

  • Laser wavelength: 658 nm
  • Scattering angle: 90°
  • Temperature range: -15 to 150° C
  • Size range (radius): 0.2-2500 nm
  • Static Scattering Molecular Weight Range: 300 Da to 1 MDa (sample dependent – maximum molecular radius 12 nm)

Probe:

  • minimum concentration: 0.1 mg/ml (14 kDa lysozyme in PBS, 100 s acquisition)
  • minimum sample volume: 1.25 μL

Time for 1 analysis (spectrum): 5-30 min.

Microscale Thermophoresis (MST)

Monolith NT.115 G009 (NanoTemper Technologies GmbH)  Applications:

Instrument: Monolith NT.115 G009 (NanoTemper Technologies GmbH)

Applications:

  • Macromolecular binding

Major Specifications:

  • Range: 200-950 nm (excitation and emission)
  • Light sources: a) LED with excitation wavelength 470 nm; b) LED with excitation wavelength 625 nm
  • IR-laser: wavelength 1475+/- 15 nm; power 120 mW max.
  • Temperature control: 20 to 50°C, accuracy +/- 0.3°C
  • KD range: 1 nM – 500 mM
  • Molecular weight range: 300 Da – 2.5 MDa

Probe:

  • One of binding partners should be labeled with a fluorescent dye
  • The experimental set: 12 – 16 glass capillaries with premixed assay solutions
  • Capillary volume: 5 μL
  • Concentration of the labeled compound: close to expected KD value or lower.

Time for 1 analysis (spectrum): Around 15 min.

Differential Scanning Fluorimetry (DSF)

Prometheus NT.48 (PR001) with Back-reflection Optics (NanoTemper Technologies GmbH)

Instrument: Prometheus NT.48 (PR001) with Back-reflection Optics (NanoTemper Technologies GmbH)

Applications:

  • Ultra-high resolution protein stability and aggregation

Major Specifications:

  • Light source: LED with excitation wavelength 285 nm
  • Fluorescence detection: 330 and 350 nm
  • Native DSF: no dye is required; tyrosine and tryptophan fluorescence is analyzed
  • Temperature range: from 15 °C to 95 °C
  • Run-by-run reproducibility of experiments in thermal ramp of 1 °C/min: +/- 0.01 °C (at 65 °C)
  • High throughput: 48 samples at a time

Probe:

  • Protein concentration range: 5 μg/ml to 250 mg/ml (standard IgG)
  • Sample volume: 10 μL

Time for 1 analysis (sassay): 1-2.5 hours

UV-Vis Spectrophotometry (UV-Vis)

UV/Vis Photodiode Array Spectrophotometer Lambda 465 (PerkinElmer)

Instrument: UV/Vis Photodiode Array Spectrophotometer Lambda 465 (PerkinElmer) with thermal scanning option

Applications:

  • Characterization of absorbance of light by biomolecules in solution in ultraviolet and visible region of spectra
  • Quantification of nucleic acids, proteins and other biomolecules
  • DNA denaturation studies
  • Enzyme kinetics

Major Specifications:

  • Wavelength range: 190-1100 nm
  • Absorbance range: ± 3 A
  • Bandwidth: 1 nm
  • Wavelength accuracy: - ± 0.2 nm
  • Wavelength reproducibility: < 0.02 nm
  • Photometric accuracy: ± 0.005 A
  • Photometric reproducibility: < 0.001 A
  • Photometric drift: < 0.001 A/hour
  • Time for full spectra acquisition: 20 msec
  • Source: deuterium and tungsten lamps

Temperature control:

  • Multi Peltier Temperature Control Unit, accommodating for up to 8 samples for automated measurement
  • Temperature control range: -10 to 100° C
  • Temperature accuracy: ± 0.15° C
  • Temperature control precision: 0.1° C

Probe:

  • minimum sample volume: 2 μL

Software: UV Lab Bio Analy Software; UV Lab Ultra Kinetics SW