Bindley Bioscience Center

Sample Prep Content

Protocols for Analytical Ultracentriguation Sample Preparation:

absorbance profile
Image taken from:
http://www.cauma.uthscsa.edu/reductants/reductants.php
  1. Provide recipe of buffer used and protein sequence to lpaul@purdue.edu
  2. Prior to the analytical ultracentrifuge runs, the sample MUST be dialyzed overnight. Submitted sample volume should be 500 μl. The last dialysis buffer will be your reference for the experiments. Please provide
    15 ml of dialysis buffer.
  3. For accurate determination of molecular weight, oligomeric state and interactions, provide three (3) different concentrations for each sample. For example a concentration series is 0.25 mg/mL, 0.50 mg/mL, and 1.00 mg/mL. Ensure that the absorbance at 280nm (or, whatever wavelength desired) is not less than 0.25 at the lowest concentration, and does not exceed 1.00 at the highest concentration.
  4. Interference optics along with absorbance at 280 nm will be used to collect the data. The interference optics is extremely sensitive to mismatched buffers.
  5. DTT, 2-mercaptoethanol, protease inhibitors and detergents will interfere with the analysis, please try to avoid. If DTT is absolutely required to maintain sample stability, then do not exceed ~ 0.5 - 1 mM concentration. When detergents and lipids are needed, please contact me before you bring in the samples.
  6. Samples will be stored at 4C and run at 20C, unless otherwise specified.

A general sample preparation protocol from Dr. Peter Schuck's Lab is available here: Download sample prep PDF

Protocol for Mass Spectometry Sample Preparation:

sample data
  1. Along with the AUC experiments, the samples will also undergo mass spectrometric analysis to check the purity of the sample along with an avg.
    molecular weight.
  2. For non-covalent analysis, please provide the sample at 1 mg/ml (~10 ul) in 25 mM Ammonium Bicarbonate, pH 9.0. (When made fresh, Ammonium Bicarbonate usually has a pH around 9.0).
  3. The mass spectrometry results will be provided along with the analytical ultracentrifugation analysis. Mass spectrometry analysis will be charged at the Purdue Proteomics Facility rates.
  4. Each sample is different and if you want to look at membrane proteins or intact non covalent interactions, then a meeting to address the question will be required.

Contact

Address
Dr. Lake Paul
Bindley Bioscience Center
Discovery Park
Purdue University
West Lafayette, IN 47907-1971