Quantifying KRAS Protein Levels in CSC-Derived Xenografts

Cytosolic proteins were isolated from an aliquot of frozen pulverized xenograft tumors used for miRNA- 126 real-time qRT-PCR analysis. Western blot analysis was done as described 63 (link) using equal amounts of cytosolic protein extract (30 µg) isolated from MDA-MB-231 and Panc1 CSC-derived xenograft tumors from test and control tumors. Cytosolic extracts were prepared by tissue homogenization in buffer containing 20 mM Tris-HCl pH 7.4, 1 mM EDTA, 250 mM sucrose, protease inhibitors, followed by centrifugation (11000g x 10 min) and final collection of supernatant (cytosolic fraction). Proteins were separated on a 15% SDS-PAGE and were transferred to PVDF membrane (Bio-Rad). The Western blot was reacted sequentially with anti-KRAS (Abcam cat# ab55391 at 20 µg/mL) followed by anti-b-actin (Santa Cruz cat# sc-47778 at 1 µg/mL) antibodies. Immunoreactive proteins were detected by chemiluminescence using the ECL Western Detection kit (GE Healthcare).

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Whitaker R.D., Decano J.L., Gormley C., Beigie C.A., Meisel C., Tan G.A., Moran A.M., Giordano N.J., Park Y., Huang P., Andersson S., Gantz D., Grant A.K., Ruiz-Opazo N., Herrera V.L, & Wong J.Y. (2022). Janus USPION modular platform (JUMP) for theranostic ultrasound-mediated targeted intratumoral microvascular imaging and DNA/miRNA delivery. Theranostics, 12(18), 7646-7667.

Publication 2022

PVDF membrane anti-b-actin ECL Western Detection kit

Actin Antibodies Buffer Cat 1 Centrifugation Chemiluminescence Cytosolic Edta Frozen Kras Membrane Mirna Protease inhibitors Proteins Pvdf Real time pcr analysis Sds page Sucrose Tissue Tris Tumors Western blot Western blot analysis Xenograft

Corresponding Organization :

Other organizations : Boston University, Beth Israel Deaconess Medical Center, Harvard University

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Variable analysis

independent variables

CSC-derived xenograft tumors from test and control tumors

dependent variables

KRAS protein expression

control variables

Cytosolic proteins isolated from MDA-MB-231 and Panc1 cell lines
Equal amounts of cytosolic protein extract (30 µg) used for Western blot analysis
Cytosolic extracts prepared by tissue homogenization in buffer containing 20 mM Tris-HCl pH 7.4, 1 mM EDTA, 250 mM sucrose, protease inhibitors, followed by centrifugation (11000g x 10 min) and final collection of supernatant (cytosolic fraction)
Proteins separated on a 15% SDS-PAGE and transferred to PVDF membrane
Western blot reacted with anti-KRAS and anti-β-actin antibodies
Immunoreactive proteins detected by chemiluminescence using the ECL Western Detection kit

controls

Positive control: MDA-MB-231 and Panc1 cell lines
Negative control: Not explicitly mentioned

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