Western Blot Analysis of AKT1

Cells were seeded in 6-well-plates (2.5 x 10⁵/well) and allowed to adhere for 24h. Whole-cell lysates were prepared using RIPA lysis-buffer containing protease inhibitor (1x, Sigma-Aldrich, St. Louis, Missouri, USA) and phosphatase inhibitor (1x, Merck Millipore, Darmstadt, Germany) and analyzed by Western blot as previously described (22 (link)). The primary antibody rabbit anti-AKT1 (#2938, Cell Signaling Technology) was used for specific AKT1-detection and the mouse anti-β-Actin (sc-47778, Santa Cruz Biotechnology) was used as loading control. Immunocomplexes were detected using peroxidase-conjugated mouse anti-rabbit IgG (sc-2357, 1:8000, Santa Cruz Biotechnology) and goat anti-mouse IgG (1030-05, 1:8000, Southern Biotech) followed by chemiluminescence detection (Westar Nova 2.0, Cyanagen Srl, Bologna, Italy) and Fuji Medical X-Ray Film (Fuji-film Corporation).

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Kempska J., Oliveira-Ferrer L., Grottke A., Qi M., Alawi M., Meyer F., Borgmann K., Hamester F., Eylmann K., Rossberg M., Smit D.J., Jücker M., Laakmann E., Witzel I., Schmalfeldt B., Müller V, & Legler K. (2023). Impact of AKT1 on cell invasion and radiosensitivity in a triple negative breast cancer cell line developing brain metastasis. Frontiers in Oncology, 13, 1129682.

Publication 2023

protease inhibitor phosphatase inhibitor rabbit anti-AKT1 mouse anti-β-Actin goat anti-mouse IgG Westar Nova 2.0 Fuji Medical X-Ray Film

Actin Akt1 Anti igg Antibody Buffer Cell Chemiluminescence Goat Mouse Peroxidase Phosphatase Protease inhibitor Rabbit Ripa Western blot X ray film

Corresponding Organization : Universität Hamburg

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Variable analysis

independent variables

Cell seeding density (2.5 x 10^5 cells/well)

dependent variables

AKT1 protein expression

control variables

Incubation time (24 hours)
Lysis buffer (RIPA buffer with protease and phosphatase inhibitors)
Primary antibodies (rabbit anti-AKT1, mouse anti-β-Actin)
Secondary antibodies (peroxidase-conjugated mouse anti-rabbit IgG, goat anti-mouse IgG)
Detection method (chemiluminescence and X-ray film)

controls

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

Annotations

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