Proteins were extracted using standard procedures in the presence of protease inhibitors57 (link). Protein lysates were fractionated by SDS-PAGE and transferred to PVDF membranes (Millipore) using TransFi (ThermoFisher Scientific). Membranes were probed for FLI1 with a mouse monoclonal antibody (1/400; BD Pharmigen). GAPDH was used as a loading control (1/200; Abcam). Secondary antibodies were HRP-conjugated with a goat anti-mouse IgG (1/1000; Abcam) and blots were developed with the ECL reagent (GE Healthcare) and exposed to film (Kodak).
Martinez-Lage M., Torres-Ruiz R., Puig-Serra P., Moreno-Gaona P., Martin M.C., Moya F.J., Quintana-Bustamante O., Garcia-Silva S., Carcaboso A.M., Petazzi P., Bueno C., Mora J., Peinado H., Segovia J.C., Menendez P, & Rodriguez-Perales S. (2020). In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells. Nature Communications, 11, 5060.
Anti iggAntibodies GapdhGoat Membranes Monoclonal antibody Mouse Protease Protein Pvdf Sds page
Corresponding Organization :
Other organizations :
Spanish National Cancer Research Centre, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Centre for Biomedical Network Research on Rare Diseases, Josep Carreras Leukaemia Research Institute, Universitat de Barcelona
Protein extraction method (standard procedures in the presence of protease inhibitors)
dependent variables
FLI1 protein expression
GAPDH protein expression (as a loading control)
control variables
SDS-PAGE fractionation and transfer to PVDF membranes
Antibodies used (mouse monoclonal anti-FLI1, anti-GAPDH, and goat anti-mouse IgG secondary)
ECL reagent used for blot development
controls
GAPDH was used as a loading control
Annotations
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