Protein Separation and Immunoblotting

Proteins (15 μg) were separated by electrophoresis on denaturing 12.5% polyacrylamide slab gels (SDS-PAGE) and transferred to PVDF membrane (IPVH00010, Immobilon P, Millipore, MA, USA), as previously described (19 (link)). Samples on the membrane were visualized by staining with Ponceau Red (Sigma-Aldrich) for 5 min. Membranes were immune-stained with rabbit polyclonal anti-H1° antibodies (1:500, sc-67324 Santa Cruz), mouse monoclonal anti-Hsc70 antibodies (1:1,000, sc-7298, Santa Cruz), rabbit polyclonal anti-SUMO1 (1:500, S373B, Santa Cruz). The secondary antibodies were AP-conjugated anti-mouse (1:7,500, S372B) and anti-rabbit (1:7,500, S373B) IgGs (Promega Corp., Madison, WI, USA).

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Schiera G., Di Liegro C.M., Puleo V., Colletta O., Fricano A., Cancemi P., Di Cara G, & Di Liegro I. (2016). Extracellular vesicles shed by melanoma cells contain a modified form of H1.0 linker histone and H1.0 mRNA-binding proteins. International Journal of Oncology, 49(5), 1807-1814.

Publication 2016

Ponceau Red sc-67324 sc-7298 AP-conjugated anti-mouse

Anti antibodies Antibodies Electrophoresis Immobilon p Membranes Monoclonal antibodies Mouse Polyacrylamide gels Promega Proteins Pvdf Rabbit Sds page Sumo1

Corresponding Organization :

Other organizations : La Maddalena, University of Palermo

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Variable analysis

independent variables

Protein samples

dependent variables

Protein separation and visualization

control variables

Denaturing 12.5% polyacrylamide slab gels (SDS-PAGE)
PVDF membrane (IPVH00010, Immobilon P, Millipore, MA, USA)
Ponceau Red staining
Primary antibodies: rabbit polyclonal anti-H1° (1:500), mouse monoclonal anti-Hsc70 (1:1,000), rabbit polyclonal anti-SUMO1 (1:500)
Secondary antibodies: AP-conjugated anti-mouse (1:7,500) and anti-rabbit (1:7,500) IgGs

controls

Positive control: Not specified
Negative control: Not specified

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