Sample: Ubiquitin CN in Hepes

Ubiquitin [13C 15N-labeled (5mgX2), non-tagged]

Sample code: BS7005UB1

Sample preparation:

• 5 mg Ubiquitin powder was dissolved in 1ml of 20mM HEPES at pH 6.8 and ~ 0.1mM DSS. Sodium-azide powder was added to prevent microbial growth.
• Note: The powder showed poor solubility and complete dissolution was not possible even after pipette mixing gently, several times.
• The sample was centrifuged for 1min @ high speed to settle the undissolved part and the clear supernatant was transferred to a 5mm NMR tube to be used for NMR experiments.
• Nanodrop was used to measure the sample concn ~ 0.03mg/ml ~ 21uM of Ubiquitin in HEPES.
• For later experiments, 2% glycerol-d8 (deuterated) was added to the sample.

Sample conditions: 0.02mM, 15N, 13C-labeled Ubiquitin in 20mM HEPES at pH 6.8 with 10% D2O and some sodium-azide and DSS powder.

1D 1H of sample at 298K ( 25 deg)

Chemical formula of buffer and additives Sample in 5mm NMR tube

Ionic Strength of the sample

Proton pulse widths are sensitive to sample ionic strength and should be calibrated for each sample. For the Ubiquitin sample in 20mM HEPES the Ionic strength was calculated by the formula below:

References:

1. Paul L. Weber, Stephen C. Brown and Lucian0 Mueller. Sequential 1H NMR Assignments and Secondary Structure Identification of Human Ubiquitin. Biochemistry 1987, 26, 7282-7290.

2.