Western Blot Protein Analysis Protocol

Whole cell lysates or nuclear proteins extracted using a nuclear extraction kit (Thermo Fisher Scientific Inc.) were prepared as detailed previously [30 (link)]. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The following primary antibodies were used for immunoblot analysis: α-SMA (ab5694), FOXM1 (ab180710), SOX9 (ab5535) from Abcam (Cambridge, MA, USA); gankyrin (12985S), Lamin B1 (13435S), phospho-AKT (9271S), PTEN (9188S) from Cell Signaling Technology (Danvers, MA); AKT (07-416) from Millipore (Burlington, MA); β-Actin (sc-1615), CK-7 (sc-70936), CK-19 (sc-33119), HNF4α (sc-6556), YAP (sc-101199) from Santa Cruz Biotechnology (Santa Cruz, CA). Membranes were blotted with primary antibody overnight at 4°C at a dilution of 1:1000 for all primary antibodies. Horseradish peroxidase–conjugated secondary antibodies for rabbit (HAF008; 1:3000) and goat (SC-2020; 1:3000) were obtained from R&D Systems (Minneapolis, MN) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Proteins were visualized with enhanced chemiluminescence reagents (ECL/ECL Plus, Amersham GE) and Kodak X-OMAT film.

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Rizvi S., Fischbach S.R., Bronk S.F., Hirsova P., Krishnan A., Dhanasekaran R., Smadbeck J.B., Smoot R.L., Vasmatzis G, & Gores G.J. (2017). YAP-associated chromosomal instability and cholangiocarcinoma in mice. Oncotarget, 9(5), 5892-5905.

Publication 2017

nuclear extraction kit HAF008 X-OMAT film

Actin Antibodies Antibody Cell Chemiluminescence Ck 19 Dilution Gankyrin Goat Horseradish peroxidase Immunoblot analysis Lamin b1 Membranes Nitrocellulose Nuclear proteins Proteins Pten Rabbit Sds page Sox9

Corresponding Organization : Mayo Clinic in Arizona

Other organizations : Charles University, University Hospital Hradec Králové, Stanford University, Mayo Clinic

Top 5 similar protocols

Variable analysis

independent variables

Whole cell lysates or nuclear proteins extracted using a nuclear extraction kit (Thermo Fisher Scientific Inc.)

dependent variables

α-SMA
FOXM1
Gankyrin
Lamin B1
Phospho-AKT
β-Actin
CK-19
HNF4α

control variables

Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes.
Membranes were blotted with primary antibody overnight at 4°C at a dilution of 1:1000 for all primary antibodies.
Horseradish peroxidase–conjugated secondary antibodies for rabbit (HAF008; 1:3000) and goat (SC-2020; 1:3000) were used.
Proteins were visualized with enhanced chemiluminescence reagents (ECL/ECL Plus, Amersham GE) and Kodak X-OMAT film.

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