Loading...
Please wait, while we are loading the content...
Similar Documents
Coupling high throughput microfluidics and small-angle x-ray scattering to study protein crystallization from solution
| Content Provider | Hyper Articles en Ligne (HAL) |
|---|---|
| Author | Pham, Van Nhat Radajewski, Dimitri Round, Adam Brennich, Martha Pernot, Petra Biscans, Béatrice Bonneté, Françoise Teychené, Sébastien |
| Abstract | In this work, we propose the combination of small-angle X-ray scattering (SAXS) and high throughput, droplet based microfluidics as a powerful tool to investigate macromolecular interactions, directly related to protein solubility. For this purpose, a robust and low cost microfluidic platform was fabricated for achieving the mixing of proteins, crystallization reagents, and buffer in nanoliter volumes and the subsequent generation of nanodroplets by means of a two phase flow. The protein samples are compartmentalized inside droplets, each one acting as an isolated microreactor. Hence their physicochemical conditions (concentration, pH, etc.) can be finely tuned without cross-contamination, allowing the screening of a huge number of saturation conditions with a small amount of biological material. The droplet flow is synchronized with synchrotron radiation SAXS measurements to probe protein interactions while minimizing radiation damage. To this end, the experimental setup was tested with rasburicase (known to be very sensitive to denaturation), proving the structural stability of the protein in the droplets and the absence of radiation damage. Subsequently weak interaction variations as a function of protein saturation was studied for the model protein lysozime. The second virial coefficients (A2) were determined from the X-ray structure factors extrapolated to the origin. A2 obtained values were found to be in good agreement with data previously reported in literature but using only a few milligrams of protein. The experimental results presented here highlight the interest and convenience of using this methodology as a promising and potential candidate for studying protein interactions for the construction of phase diagrams. |
| Ending Page | 2287 |
| Page Count | 6 |
| Starting Page | 2282 |
| File Format | |
| ISSN | 00032700 |
| e-ISSN | 15206882 |
| DOI | 10.1021/acs.analchem.6b03492 |
| Journal | Analytical Chemistry |
| Issue Number | 4 |
| Volume Number | 89 |
| Language | English |
| Publisher | American Chemical Society |
| Publisher Date | 2017-01-17 |
| Access Restriction | Open |
| Subject Keyword | Crystallization Small Angle X Ray Scattering Microfluidics Mathematical Models Droplets Proteins Saturation Nanostructure chim spi Chemical Sciences Chemical engineering Engineering Sciences [physics] Chemical and Process Engineering |
| Content Type | Text |
| Resource Type | Article |
| Subject | Analytical Chemistry |
