Journal: Journal of materials chemistry. B

Article Title: Attenuation of Thrombin-Mediated Fibrin Formation via Changes in Fibrinogen Conformation Induced by Reaction with S- nitroso- N -acetylpenicillamine, but not S- nitrosoglutathione

doi: 10.1039/C8TB02103A

Figure Lengend Snippet: Western immunoblot analysis of fibrinogen subunits, Aa, Bb and γ after 1 h exposure with SNAP, NAP and SNP in absence and presence of thrombin. (A), Representative immunoblots. Aliquots (15 μl) of samples were separated on 12% polyacrylamide gels, protein transferred to poly(vinyl-difluoride) (PVDF) membranes and then immunostained with mouse anti-human alpha fibrinogen, rabbit anti-human beta fibrinogen and goat anti-human gamma fibrinogen antibodies (all diluted 1:200). After membrane washings, the membranes were incubated with the secondary antibodies chicken anti-mouse-AF488, chicken anti-rabbit-AF647 and donkey anti-goat-AF555 (all diluted 1:500) for 1 h. After two 10 min washes the blots were imaged in a fluorescence imager (Bio-Rad VersaDoc MP4000 Hercules, CA). Molecular weight markers are shown on left with positions of the relevant fibrinogen chains and Aαγ dimers indicated on the right. (B) Western analysis of samples in (A), after the 1 h incubation, were quantified by densitometry using Quanity One imaging software (Bio-Rad Hercules, CA). The specific Aαγ dimer formation was calculated as the ratio (density) of the Aαγ dimer band over the sum of dimers plus monomers. The data was collected after 1 h in presence of thrombin. The ratio data are means + SEM (n=3). * = p<0.05, SNAP or NAP vs SNP.

Article Snippet: After membrane washings, the membranes were incubated with the secondary antibodies chicken anti-mouse-AF488, chicken anti-rabbit-AF647 and donkey anti-goat-AF555 (all diluted 1:500) for 1 h. After two 10 min washes the blots were imaged in a fluorescence imager (Bio-Rad VersaDoc MP4000 Hercules, CA).

Techniques: Western Blot, Incubation, Fluorescence, Molecular Weight, Imaging, Software