Whole cell lysates were made using denaturing lysis buffer containing protease/phosphatase inhibitors (Cell Signaling Technology 5872). Cells were kept on ice and lysed for 30 min. Samples were then sonicated for 5 sec and spun at max speed in a microcentrifuge for 15 min at 4°C, after which supernatant was collected into a clean tube. Protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific 23227) and 30 μg protein was then loaded on 4–12% Tris-Glycine gels, transferred to PVDF membrane, and probed with antibodies in 5% milk in TBST. Primary antibodies used include: Actin Beta (Santa Cruz Biotechnology sc-47778, RRID:AB_626632) used at 1:2500 and TBX2 (Santa Cruz Biotechnology sc-514291, RRID:AB_2941848) used at 1:250. The secondary antibody used was Goat anti-mouse HRP (Thermo Fisher 31430, RRID:AB_228307) at 1:25,000. Blots were developed using SuperSignal West Femto Maximium Sensitivity Substrate (Thermo Fisher Scientific 23227) on the Chemidoc Imaging System (Biorad).
Supersignal west femto maximium sensitivity substrate
Manufactured by Thermo Fisher Scientific
SuperSignal West Femto Maximium Sensitivity Substrate is a chemiluminescent substrate designed for detecting low-abundance proteins in Western blot analysis. It is capable of detecting femtogram levels of target proteins.
Automatically generated - may contain errors
2 protocols using supersignal west femto maximium sensitivity substrate
1
Whole Cell Lysate Preparation and Western Blot
2
Western Blot Analysis of TBX2
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Whole cell lysates were made using denaturing lysis buffer containing protease/phosphatase inhibitors (Cell Signaling Technology 5872). Cells were kept on ice and lysed for 30 min. Samples were then sonicated for 5 sec and spun at max speed in a microcentrifuge for 15 min at 4°C, after which supernatant was collected into a clean tube. Protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific 23227) and 30 μg protein was then loaded on 4-12% Tris-Glycine gels, transferred to PVDF membrane, and probed with antibodies in 5% milk in TBST. Primary antibodies used include: Actin Beta (Santa Cruz Biotechnology sc-47778, RRID:AB_626632) used at 1:2500 and TBX2 (Santa Cruz Biotechnology sc-514291, RRID:AB_2941848) used at 1:250. The secondary antibody used was Goat anti-mouse HRP (Thermo Fisher 31430, RRID:AB_228307) at 1:25,000. Blots were developed using SuperSignal West Femto Maximium Sensitivity Substrate (Thermo Fisher Scientific 23227) on the Chemidoc Imaging System (Biorad).
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