Clarity max western ecl substrate kit

Western blot analysis was carried out as described earlier [13 (link)]. Briefly, 4T1-Qt cells were lysed using RIPA buffer, and the resulting lysate was precipitated (15 min, 14,000× g). Sample buffer 5× was added to different amounts of cell lysate, heated at 95 °C, then cooled on ice. Samples were loaded onto a gel and electrophoresed (80 V for 25 min and 100 V for 1.5 h); then, the gel was transferred into a transfer buffer. The nitrocellulose membrane was activated and placed over the gel. The transfer was carried out in a chamber filled with transfer buffer for 1 h at 100 V. Then, the membrane was washed three times to remove transfer buffer residues in PBST. To prevent nonspecific binding, the membrane was incubated in a PBST solution with 5% non-fat milk for 2 h and washed again. The membrane was incubated with anti-DYKDDDDK Tag antibodies (1:1000, BioLegend, San Diego, CA, USA) for 2 h, followed by washing three times. After that, alkaline horseradish peroxidase conjugated secondary antibodies (1:1000, goat anti-mouse IgG, Santa Cruz Biotechnology,) were added. Clarity Max Western ECL Substrate kit (BioRad, Hercules, CA, USA) was used to reveal the result. The results were registered with the ChemidocMP Imaging system (BioRad, Hercules, CA, USA).

Protein samples separated on SDS-PAGE gels were transferred to the polyvinylidene fluoride (PVDF) membrane. The semi-dry transfer was performed in Tris-glycine buffer with SDS and isopropanol using Biometra Flashblot (Analytik Jena GmbH). Parameters of the analysis were set to 15 V, 250 mA for 45 min. Subsequently, the PVDF membrane was incubated in specific antibody solution (mouse anti-His) (Invitrogen) with dilution of 1:1000 in a 1% skim milk solution at 4 °C overnight. Further, the membrane was washed in goat anti-mouse secondary antibody (1:10 000) in 1% skim milk solution (Goat Anti-Mouse IgG, Sigma Aldrich) for 1 h at room temperature. For signal visualisation, the membrane was washed in chemiluminescence solution using Clarity Max Western ECL Substrate kit (Bio-Rad) according to the protocol and signal was detected using ImageQuant LAS 500 chemiluminescence CCD camera (GE Healthcare).

The cells were lysed with RIPA buffer, and the lysates were centrifuged (15 min, 14,000× g). Buffer for probes (5×) was added to 2, 5, 10, and 20 µL of cell lysate, and the samples were heated to 95 °C for 5 min and then cooled down by placing on ice. The samples were put in the gel, and electrophoresis was run on 80 V for 25 min and 100 V for 90 min. PageRuler-prestained protein ladder (Thermo Fisher) served as a length marker. The gel was placed to transfer buffer (25 mM Tris buffer, 192 mM glycine, pH 8.3, 10% ethanol, 0.25% SDS). Nitrocellulose membrane activated with 96% ethanol was placed over the gel. The samples transfer was performed in a bath with transfer buffer on 100 V for 1 h. Afterward, the membrane was washed 3 times from residues of transfer buffer in PBS-0.1% Tween (PBST). To block unspecific protein binding, the membrane was incubated in PBST supplemented with 5% skimmed milk for 2 h, then washed again. The membrane was incubated with primary antibodies against FLAG-tag (Sigma-Aldrich, 1:1000) for 2 h and then washed 3 times. Secondary goat anti-mouse IgG antibodies (Santa Cruz Biotechnology, 1:1000) conjugated with alkaline horseradish peroxidase. Clarity Max Western ECL Substrate kit (BioRad) was used to reveal the result according to the manufacturer’s protocol. The result was registered with the ChemidocMP Imaging system (BioRad).