Western Blot Quantification of Protein Markers

Thirty micrograms of proteins, acquired from all samples, were processed as earlier mentioned (Mammana et al., 2019 (link)). Membranes were incubated with primary antibodies rabbit anti-VEGF (1:750; rabbit; Sigma-Aldrich, Milan, Italy), RUNX2 (1:750; rabbit; Sigma-Aldrich), and anti-beta-actin (1:750; mouse; Santa Cruz Biotechnology, Santa Cruz, CA, United States). After five washes in PBS containing 0.1% Tween-20, samples were incubated for 1 h at room temperature with peroxydase-conjugated secondary antibody anti-rabbit and anti-mouse diluted 1:2,000 in 1 × PBS, 3% milk, 0.1% Tween (Mazzatenta et al., 2014 (link)). Protein expression was detected using the enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech, Milan, Italy) with photo documenter Alliance 2.7 (Uvitec, Cambridge, United Kingdom). Signals were captured by ECL enhancing and analyzed using a UVIband-1D gel analysis (Uvitec).

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Marconi G.D., Diomede F., Pizzicannella J., Fonticoli L., Merciaro I., Pierdomenico S.D., Mazzon E., Piattelli A, & Trubiani O. (2020). Enhanced VEGF/VEGF-R and RUNX2 Expression in Human Periodontal Ligament Stem Cells Cultured on Sandblasted/Etched Titanium Disk. Frontiers in Cell and Developmental Biology, 8, 315.

Publication 2020

rabbit anti-VEGF RUNX2 anti-beta-actin Alliance 2.7 UVIband-1D gel analysis

Anti antibodies Antibody Beta actin Chemiluminescence Membranes Milk Mouse Protein Rabbit Runx2 Tween Tween 20 Vegf

Corresponding Organization :

Other organizations : University of Chieti-Pescara, Ospedale SS. Annunziata, Centro Neurolesi Bonino Pulejo

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Variable analysis

independent variables

Thirty micrograms of proteins, acquired from all samples

dependent variables

Protein expression of VEGF, RUNX2, and beta-actin

control variables

Processing of samples as earlier mentioned (Mammana et al., 2019)
Incubation of membranes with primary antibodies
Five washes in PBS containing 0.1% Tween-20
Incubation with peroxydase-conjugated secondary antibody
Protein expression detection using enhanced chemiluminescence detection system (ECL)

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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