Western Blot Analysis of Myostatin, Oct4, and PCNA

Immuno-detection on the membranes was with primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) against myostatin (GDF8/11, mouse monoclonal antibody SC-393335); and housekeeping beta-actin (mouse monoclonal, followed by secondary antibodies: anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology, Danvers, MA, USA), or anti-rabbit IgG linked to HRP (Amersham GE, Pittsburgh, PA, USA). Oct4 was estimated by a polyclonal antibody from Biovision catalog 3576, at 1:500; and PCNA with a monoclonal antibody from Millipore MAB424, at 1:2000. Bands were visualized using luminol (SuperSignal West Pico; Chemiluminescent, Pierce, Rockford, IL, USA). For negative controls, the primary antibody was omitted. Densitometric analysis was performed in certain cases, as stated, correcting by the housekeeping proteins [5 (link),7 (link),8 (link)].

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Kovanecz I., Gelfand R., Sharifzad S., Ohanian A., DeCastro W.B., Cooper C., Lin G., Lue T, & Gonzalez-Cadavid N.F. (2020). Evaluation of the In Vitro Damage Caused by Lipid Factors on Stem Cells from a Female Rat Model of Type 2 Diabetes/Obesity and Stress Urinary Incontinence. International Journal of Molecular Sciences, 21(14), 5045.

Publication 2020

SC-393335 horseradish peroxidase (HRP)-linked antibody MAB424

Anti igg Antibodies Antibody Beta actin Densitometric Gdf8 Horseradish peroxidase Luminol Membranes Monoclonal antibody Mouse Oct4 Pcna Proteins Rabbit

Corresponding Organization : Charles R. Drew University of Medicine and Science

Other organizations : University of California, San Francisco

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Variable analysis

independent variables

Primary antibodies against myostatin (GDF8/11, mouse monoclonal antibody SC-393335)
Primary antibodies against housekeeping beta-actin (mouse monoclonal)
Primary antibody against Oct4 (polyclonal antibody from Biovision catalog 3576, at 1:500)
Primary antibody against PCNA (monoclonal antibody from Millipore MAB424, at 1:2000)

dependent variables

Immunodetection on the membranes

control variables

Secondary antibodies: anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology, Danvers, MA, USA), or anti-rabbit IgG linked to HRP (Amersham GE, Pittsburgh, PA, USA)

controls

Positive control: Presence of primary antibodies
Negative control: Omission of primary antibody

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