UV-Visible Spectroscopy for Monitoring Fibrin Clot Formation
Published: June 29, 2023
Abstract
Source: Singh, P. K. et al., Analysis of β-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy. J. Vis. Exp. (2018)
This video demonstrates the use of a UV-visible spectrophotometer for monitoring insoluble fibrin clot formation as a function of the change in the solution turbidity. The UV-visible spectrophotometer measures the solution’s absorbance over time, which represents the turbidity change due to the insoluble fibrin clot formation.
Protocol
1. Preparation of Aβ42 and Fibrinogen for Analysis Prepare monomeric Aβ42 from lyophilized powder Warm Aβ42 powder to room temperature and spin down at 1,500 x g for 30 s. Add 100 µL of ice-cold hexafluoroisopropanol (HFIP) per 0.5 mg of Aβ42 powder and incubate for 30 min on ice. CAUTION: Use care when handling HFIP and perform all steps in a chemical hood. Prepare 20 µL aliquots and let the films air-dr…
Divulgations
The authors have nothing to disclose.
Materials
| Fibriogen, Plasminogen-Depleted, Plasma | EMD Millipore | 341578 | Keep lid parafilm wrapped to avoid exposure to moisture |
| Beta-Amyloid (1-42), Human | Anaspec | AS-20276 | |
| Thrombin from human plasma | Sigma-Aldrich | T7009 | |
| 1,1,1,3,3,3-Hexafluoro-2-propanol, Greater Than or Equal to 99% | Sigma-Aldrich | 105228 | |
| Dimethyl sulfoxide (DMSO), sterile-filtered | Sigma-Aldrich | D2438 | |
| Pierce BCA Protein Assay Kit | Thermo Scientific | 23225 | |
| Tris Base | Fischer Scientific | BP152 | |
| HEPES | Fischer Scientific | BP310 | |
| NaCl | Fischer Scientific | S271 | |
| CaCl2 | Fischer Scientific | C70 | |
| Filter Syringe, 0.2µM, 25mm | Pall | 4612 | |
| Millex Sterile Syringe Filters, 0.1 um, PVDF, 33 mm dia. | Millipore | SLVV033RS | |
| Solid 96-Well Plates High Binding Certified Flat Bottom | Fischer Scientific | 21377203 | |
| Spectramax Plus384 | Molecular Devices | 89212-396 | |
| Centrifuge, 5417R | Eppendorf | 5417R | |
| Lab Rotator | Thermo Scientific | 2314-1CEQ |
Tags
Citer Cet Article
UV-Visible Spectroscopy for Monitoring Fibrin Clot Formation. J. Vis. Exp. (Pending Publication), e21471, doi: (2023).