UV-Visible Spectroscopy for Monitoring Fibrin Clot Formation

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…

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