Quantitative Western Blot Analysis of SHH and VEGF

Protein lysates were obtained from WJ-MSC monolayers and homogenized in lysis buffer composed of a 1× protease inhibitor mix (Thermo Scientific). Protein concentration was determined (DC™ Protein Assay; BioRad), and a 50-μg protein concentration was loaded for SDS-PAGE and blotted on 0.45-μm pore nitrocellulose membranes. Membranes were blocked and incubated with anti-SHH or vascular endothelial growth factor (VEGF) antibodies. SHH western blots were carried out as previously described [53 (link)] using a 5E1 antibody (Hybridoma supernatant concentrated from Hybridoma Bank; dilution 1/1000). VEGF was detected using rabbit anti-VEGF (Abcam; ab46154; 1/1000). Different positive control samples were used for both proteins (see Results section). Antigens were detected via chemiluminescence using ECL solutions (SuperSignal™ West Pico or Femto Maximum Sensitivity Substrate; Thermo Scientific). Exposed X-ray films (Fujifilm) were analyzed with the Relative Pixel Intensity tool from ImageJ (NIH, USA).

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Zavala G., Prieto C.P., Villanueva A.A, & Palma V. (2017). Sonic hedgehog (SHH) signaling improves the angiogenic potential of Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC). Stem Cell Research & Therapy, 8, 203.

Publication 2017

DC™ Protein Assay ab46154 SuperSignal™ West Pico Femto Maximum Sensitivity Substrate

Antibodies Antibody Antigens Assay Buffer Chemiluminescence Dilution Hybridoma Membranes Nitrocellulose Protease inhibitor 1 Proteins Rabbit Sds page Sensitivity Vascular endothelial growth factor Western blots X ray films

Corresponding Organization :

Other organizations : University of Chile

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Variable analysis

independent variables

Protein lysates obtained from WJ-MSC monolayers

dependent variables

Protein concentration
Protein expression of SHH and VEGF

control variables

Lysis buffer composed of a 1× protease inhibitor mix (Thermo Scientific)
Protein concentration loaded for SDS-PAGE (50 μg)
Nitrocellulose membrane pore size (0.45 μm)
Antibodies used: anti-SHH (5E1 antibody, Hybridoma supernatant concentrated from Hybridoma Bank, dilution 1/1000) and anti-VEGF (rabbit anti-VEGF, Abcam; ab46154, 1/1000)

positive controls

Different positive control samples used for both SHH and VEGF proteins (see Results section)

negative controls

No negative controls explicitly mentioned

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