Western Blot Analysis of Cancer Antigens

Whole protein was extracted and quantitated as previously described [50 (link)]. 30-100 μg of protein was loaded onto a NuPAGE^® Novex^® 4-12% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane (Invitrogen). 5% blotting grade blocker (Bio-Rad, Hercules, CA) in phosphate-buffered saline was used to block nonspecific binding. Membranes were incubated overnight at 4°C with NY-ESO-1 (Invitrogen, clone E978) or MAGE-A antibodies (Invitrogen, clone 6C1) at 1:200, then incubated with secondary antibody (GE Healthcare Life Sciences, Piscataway, NJ) at 1:3000 dilution for 1hr. β-actin antibody (MP Biomedicals, Santa Ana, CA, clone C4) at 1:10,000 dilution was used as a loading control. Proteins were visualized using an enhanced chemiluminescence detection kit (GE Healthcare Life Sciences). As a positive control for NY-ESO-1 expression, we used protein derived from OVCAR-3 cells treated with decitabine as previously described [51 (link)].

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Srivastava P., Paluch B.E., Matsuzaki J., James S.R., Collamat-Lai G., Blagitko-Dorfs N., Ford L.A., Naqash R., Lübbert M., Karpf A.R., Nemeth M.J, & Griffiths E.A. (2016). Induction of cancer testis antigen expression in circulating acute myeloid leukemia blasts following hypomethylating agent monotherapy. Oncotarget, 7(11), 12840-12856.

Publication 2016

NuPAGE® Novex® 4-12% Bis-Tris gel nitrocellulose membrane 5% blotting grade blocker β-actin antibody enhanced chemiluminescence detection kit

Actin Antibodies Antibody Bis tris Block Cells Chemiluminescence Clone Decitabine Dilution Membranes Nitrocellulose Phosphate Protein Saline

Corresponding Organization :

Other organizations : Roswell Park Comprehensive Cancer Center, University Medical Center Freiburg, University of Freiburg, University of Nebraska Medical Center, Nebraska Medical Center

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Variable analysis

independent variables

Protein extraction and quantitation method
Amount of protein loaded onto the gel (30-100 μg)

dependent variables

Protein expression levels of NY-ESO-1 and MAGE-A

control variables

Gel type (NuPAGE® Novex® 4-12% Bis-Tris gel)
Transfer to nitrocellulose membrane
Blocking with 5% blotting grade blocker in phosphate-buffered saline
Primary antibody dilution (1:200)
Secondary antibody dilution (1:3000)
β-actin antibody dilution (1:10,000) as a loading control

positive control

Protein derived from OVCAR-3 cells treated with decitabine

negative control

Not explicitly mentioned

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