Skip to content

JASCO JASCO

  • News
  • Events
  • E-Store
  • My Account
  • Contact Us
  • Worldwide
Search
Click to view menu
MENUMENU
  • Products
    • Chromatography
      • HPLC
      • RHPLC
      • UHPLC
      • LC-MS
      • Preparative LC
      • Analytical SFC
      • Semi-Preparative SFC
      • Hybrid SFC
      • Fuel Analysis by SFC-FID
      • Preparative SFC
      • Supercritical Fluid Extraction
      • Chromatography Software
    • Molecular Spectroscopy
      • Circular Dichroism
      • High-Throughput CD
      • Vibrational CD
      • Circularly Polarized Luminescence (CPL)
      • Polarimeters
      • FTIR Spectrometers
      • FTIR Microscopy
      • FTIR Portable
      • Raman Microscopy
      • Palmtop Raman Spectrometer
      • Probe Raman
      • UV-Visible/NIR Spectrophotometers
      • UV-Visible/NIR Microscopy
      • Fluorescence Spectrophotometers
      • Film Thickness
      • Spectra Manager™ Suite
    • Refurbished
      • Refurbished HPLC Systems
      • HPLC Switching Valves
      • NRS-4100 Confocal Raman Microscopes
      • FTIR Accessories
  • Service
    • Service and Support Plans
    • Service Request Form
  • Applications
  • KnowledgeBase
  • Learning Center
    • Best Practices
      • Circular Dichroism Tips & Tricks for Biological Samples
      • CD Scale Calibration with ACS
      • Fluorescence Tips & Tricks
      • Raman Spectroscopy Tips & Tricks
    • Training Videos
      • ChromNAV
      • SF-NAV
      • Circular Dichroism
      • UV-Visible/NIR
      • Fluorescence
    • Training Seminars
      • Registration Form
    • Webinars
    • eBooks
    • Theory
      • Theory of Molecular Spectroscopy
      • Chromatography
  • About Us
    • President’s Message
    • Contact
    • History
    • Careers
  • News
  • Events
  • Worldwide
  • Shop
  • My Account

Home / Applications / Microassay Thermal Denaturation of Ribonuclease A Probed by Circular Dichroism Spectroscopy

  • Industry

  • Technique

Microassay Thermal Denaturation of Ribonuclease A Probed by Circular Dichroism Spectroscopy

By Leah Pandiscia, PhD

image_pdfView and Download PDF
August 17, 2022

Introduction

Circular dichroism (CD) is a useful technique to measure and analyze secondary structure and the thermal denaturation of proteins and nucleic acids in the far-UV region. Typically, a 1 mm pathlength rectangular cell is used for these measurements. However, this requires a sample volume of about 200 µL. In the case of samples where only very small volumes are available, JASCO now offers a new capillary cell for sample volumes smaller than 10 µL. The capillary cell also has a jacket for thermal ramping measurements, shown in Figure 1. CD measurements remain simple to perform and the capillary cells are inexpensive and disposable.

This application note demonstrates the use of a capillary cell and jacket for thermal ramping studies of ribonuclease A.

J-1500 Circular Dichroism Spectrometer
J-1500 Circular Dichroism Spectrometer

Experimental

Measurement conditions
Data acquisition interval0.2°C
Path length0.5 mm (capillary cell), 1 mm (rectangular cell)
Spectral bandwidth1 nm
Rising temperature rate1°C/min
Measurement wavelength222 nm
Response time8 sec

1 mg/mL of ribonuclease A aqueous solution is drawn up into a capillary cell with a 0.5 mm optical pathlength, as seen in Figure 1. The cell is then inserted into the capillary jacket for thermal ramping CD measurements.

Capillary cell preparation diagram. For greater visibility of a sample in the photos above, a colored sample was used instead of ribonuclease A solution.
Figure 1. Capillary cell preparation diagram. For greater visibility of a sample in the photos above, a colored sample was used instead of ribonuclease A solution.

Keywords

Circular dichroism, capillary cell, microassay, thermal denaturation, ribonuclease A, Denatured Protein Analysis software, denaturation temperature, J-1500

Results

Figure 1 shows the thermal denaturation of ribonuclease A. The JASCO JWTDA-519 Denatured Protein Analysis software was used to calculate the denaturation temperature of ribonuclease A in both the capillary and rectangular cells. 59.4°C was calculated for the capillary cell which is in good agreement with 59.7°C obtained for the denaturation temperature in the rectangular cell.

Thermal denaturation curve of ribonuclease A measured at a wavelength of 222 nm. The green curve was obtained using the capillary cell while the blue curve was measured using the 1 mm rectangular cell.
Figure 2. Thermal denaturation curve of ribonuclease A measured at a wavelength of 222 nm. The green curve was obtained using the capillary cell while the blue curve was measured using the 1 mm rectangular cell.

Conclusion

This application note demonstrates that the denaturation temperature values, along with the thermal denaturation curves shown in Figure 2, illustrate that the microassay of ribonuclease A using the capillary cell and jacket can be carried out with high accuracy.

Note: The MSD-462 microsampling disc can be used for spectral scanning measurements on sample volumes between 2 and 10 µL. The MSD-462 applications are shown in the following Application Notes: 260-CD-0011 and 260-CD-0019.

image_pdfView and Download PDF

Featured Products:

  • Automated high-throughput screening system

    High-Throughput CD

  • J-1100 Circular Dichroism Spectrophotometer

    The basic, compact model for research, QA/QC and teaching applications.

    J-1100

  • J-1500 Circular Dichroism Spectrophotometer

    Our highest performing model with a wide range of accessories for maximum flexibility.

    J-1500

  • J-1700 Circular Dichroism Spectrophotometer

    UV, Visible, and NIR measurements up to 2500 nm for MCD and specialized applications.

    J-1700

About the Author

Leah Pandiscia received her PhD from Drexel University where she studied Biophysical Chemistry. She is a Spectroscopy Applications Scientist.

JASCO provides services and support to a diverse range of industries throughout the world.

View our support plans

Connect with JASCO

  • Facebook
  • Twitter
  • LinkedIn
  • JASCO Sales
  • 800-333-5272

Receive the latest promotions and special offers

  • Careers
  • Press Kit
  • Privacy Policy
  • Sitemap

© , JASCO. All Rights Reserved.