Laemmli buffer 2x

Mouse tissues were weighed out and ice-cold lysis buffer was added to approx. 0.2 to 0.5 mg tissue/ml buffer (10 mM K₂HPO₄, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100, 0.2% Na-deoxycholate). Fresh protease inhibitors PMSF 0.5 mM, Na₃VO₄ 0.1 mM, NaF 50mM, Jenny’s mix (SBTI 3.2 μg/ml, Aprotinin 2 μg/ml, Benzamidin 0.5 mM) were added to the buffer.
Samples were first homogenized, and then pellet debris were centrifuged at 4°C for 30 min at max speed (tabletop) and the supernatant (soluble proteins) was transferred into fresh tube (pellets = non-soluble proteins).
Protein concentrations were determined with BCA assay. Cell lysates were loaded using Laemmli Buffer 2x (Sigma Aldrich) in equal mass (see above for the immunoblot procedure) into 8% polyacrylamide gels.
The membrane was then incubated overnight at 4°C with primary antibody solution (1:1,000 anti β-actin (13E5) rabbit primary antibody #4970 from Cell Signaling) (1:1,000 anti GAPDH) (1:1,000 anti-tdTomato rabbit primary antibody #632496 from Takara).

Detection of specific proteins among the experimental series required a proper sample preparation, in which, for each specimen, the Laemmli buffer 2X (S3401; Sigma-Aldrich) was mixed with an equal protein amount before heat denaturation (5 min at 95 °C). Subsequently, loading a protein range from 15 to 30 µg, electrophoresis was performed in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). After moving proteins to the nitrocellulose membrane (Amersham Protran Premium; Cytiva, Marlborough, MA, United Stetes), foils were firstly blocked for one hour in no-fat milk (5% w/v) (A0830; PanReac Applichem, Chicago, IL, USA) and then incubated overnight at 4 °C with specific primary antibodies. The next morning, HRP secondary antibodies, recognizing the related primary species, were applied to the membrane for 1 h at room temperature. Finally, chemiluminescence was detected with the Chemi Doc XRS (Bio-Rad, Hercules, CA, USA) instrument using liteablot chemiluminescent as a substrate (EMP011005; EuroClone). Each incubation step was preceded and followed by three washes in TBS plus 0.05% Tween-20 (TC287; HIMEDIA, Mumbai, India) (T-TBS).