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

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Citer Cet Article
UV-Visible Spectroscopy for Monitoring Fibrin Clot Formation. J. Vis. Exp. (Pending Publication), e21471, doi: (2023).

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