Protein Expression Analysis by Western Blot

Thirty micrograms of proteins obtained from all samples were processed as previously described [29 (link)]. Blotted membranes were incubated with the following primary antibodies: rabbit anti-RUNX2 (1:750, rabbit; Sigma-Aldrich, Milan, Italy), anti-Vimentin (1:750, rabbit; Sigma-Aldrich), anti-Laminin (1:750, rabbit; Sigma-Aldrich), anti-N-cadherin (1:750, rabbit; Sigma-Aldrich) and anti-beta-actin (1:750, mouse; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After five washes in PBS containing 0.1% Tween-20, membranes were incubated for 1 h at RT with peroxidase-conjugated anti rabbit and anti-mouse secondary antibodies (1:2000; ThermoFisher Scientific, Milan, Italy). Protein expression was analysed by the enhanced chemiluminescence detection method (ECL) (Amersham Pharmacia Biotech, Milan, Italy) with photo documenter Alliance 2.7 (Uvitec, Cambridge, UK). Signals were evaluated by ECL enhancing and assessed through an UVIband-1D gel analysis system (Uvitec) [30 (link)].

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Marconi G.D., Fonticoli L., Della Rocca Y., Rajan T.S., Piattelli A., Trubiani O., Pizzicannella J, & Diomede F. (2021). Human Periodontal Ligament Stem Cells Response to Titanium Implant Surface: Extracellular Matrix Deposition. Biology, 10(9), 931.

Publication 2021

rabbit anti-RUNX2 anti-Vimentin anti-Laminin anti-N-cadherin anti-beta-actin Alliance 2.7

Anti antibodies Antibodies Beta actin Cadherin Chemiluminescence Laminin Membranes Mouse Peroxidase Protein Rabbit Runx2 Tween 20 Vimentin

Corresponding Organization : Ospedale SS. Annunziata

Other organizations : University of Chieti-Pescara, Karpagam Academy of Higher Education

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression of RUNX2, Vimentin, Laminin, N-cadherin, and beta-actin

control variables

Amount of protein processed (30 micrograms)
Primary antibodies used (rabbit anti-RUNX2, anti-Vimentin, anti-Laminin, anti-N-cadherin, and anti-beta-actin)
Secondary antibodies used (peroxidase-conjugated anti-rabbit and anti-mouse)
Incubation time and temperature of primary and secondary antibodies
Washing steps in PBS with 0.1% Tween-20
Detection method (enhanced chemiluminescence)

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

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