Western Blot Analysis of Cytosolic and Nuclear Proteins

The respective cytosolic or nuclear proteins were separated by SDS-PAGE and transferred to nitrocellulose filters. Filters were blocked in 5% nonfat dry milk and probed with primary antibody overnight.[22 (link)] Primary antibodies such as HO-1, Akt, phospho-Akt (Ser 473), glyceraldehyde-6-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA; whereas Bax, Bcl-2, Src and phospho-Src (Tyr416), were obtained from Cell Signaling Technology, Beverly, MA, USA. All primary antibodies were used at a dilution of 1:1000. Protein bands were identified with horseradish peroxidase conjugated secondary antibody (1:2000 dilutions) and Western blotting luminol reagent (Santa Cruz Biotechnology). GAPDH was used as loading control for cytosolic and nuclear fraction. The resulting blots were digitized, subjected to densitometric scanning using a QuantiOne software (Bio-Rad), and normalized against loading control.

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Jayachandran K.S., Vasanthi A.H, & Gurusamy N. (2016). Steroidal Saponin Diosgenin from Dioscorea bulbifera Protects Cardiac Cells from Hypoxia-reoxygenation Injury Through Modulation of Pro-survival and Pro-death Molecules. Pharmacognosy Magazine, 12(Suppl 1), S14-S20.

Publication 2016

Bax Bcl-2 phospho-Src (Tyr416), horseradish peroxidase conjugated secondary antibody Western blotting luminol reagent QuantiOne software

Antibodies Antibody Bcl 2 Cytosolic Dehydrogenase Dilutions Glyceraldehyde dehydrogenase Glyceraldehyde phosphate Horseradish peroxidase Luminol Milk Nitrocellulose Nuclear proteins Phosphate Protein Sds page

Corresponding Organization : Pondicherry University

Other organizations : SASTRA University, King Khalid University

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Variable analysis

independent variables

Primary antibodies (HO-1, Akt, phospho-Akt (Ser 473), Bax, Bcl-2, Src and phospho-Src (Tyr416))

dependent variables

Protein bands identified with horseradish peroxidase conjugated secondary antibody and Western blotting luminol reagent

control variables

GAPDH (glyceraldehyde-6-phosphate dehydrogenase) used as loading control for cytosolic and nuclear fraction

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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