Protein or RNA Secondary Structure and Thermal Stability by Circular Dichroism

The Facility’s J-1100 circular dichroism (CD) spectrometer is a powerful tool to compare the relative structure content and thermal stability of proteins and nucleic acid samples in solution. Applications include:

• Estimate the secondary structure content of proteins or nucleic acids: CD spectra have typical shapes for a-helical, b-sheet, unfolded proteins and nucleic acids.
• Measure the thermal stability of a protein or nucleic acid sample from a melting curve.
• Demonstrate similar fold and stability of protein samples that carry point mutations, which fulfills new NIH guidelines on Transparency and Reproducibility.
• Compare structure and thermal stability of phylogenetically-related, alternatively spliced, post-transcriptionally or post-translationally-modified protein samples.

Sample requirements and cost are minimal.  Dr. Jermaine Jenkins, a trained expert in CD methods, is available to consult for experiment design, use, and data analysis. Please contact Dr. Jermaine Jenkins for instrument sign-up or consultation.

Sample Requirements

• ~2 mL volume for 10 mm cell (~300 µL for 1 mm cell but harder to use/clean and BYO).
• No chloride ions in the solution; phosphate is the most commonly used counter anion.
• ~0.2 mg/mL (exact concentration depends on sample cell). For typical 20 kDa protein, this would be 10 µM concentration.

Sign-up Procedure

Adequate purging of the instrument with nitrogen gas BEFORE firing the lamp is critical to avoid damaging the optics. No safety checks are built into the software. To avoid inadvertent firing of the lamp before purging that would damage costly optics, we will ask users for the following:

1. Please sign up for use of the CD instrument using a web-based calendar, which is administered by Dr. Jenkins. Users should also please notify Dr. Jenkins about planned use of the instrument by email.
2. Half an hour before the scheduled start time, Dr. Jenkins will initiate purging the instrument.
3. Please notify Dr. Jenkins of any cancelations or time changes 10 minutes before the scheduled start or they will be charged the start-up fee.
4. The CD instrument is located in Room 3-8546 of the Dept. of Biochemistry & Biophysics.

Charges for Use are Based Solely on Running Costs

• $45 start-up fee per experiment for pre-purging and standardization of the instrument.
• $15 per hour for subsequent use, which accounts for the costs of nitrogen  and of the lamp and mirror replacements.
• $20 for use of the facility cuvette. This fee covers the time required for Dr. Jenkins to clean the cuvette and part of the charge is “banked” for covering the costs of cuvette replacement ($170/each). This charge is waived for users who bring their own cuvette, which will be encouraged (e.g., there will be no separate cuvettes for proteins and RNA).