Tgs buffer

Samples were heated for 10 minutes at > 90°C. Proteins were run on 4–20% gels (Bio-Rad Mini-PROTEAN® TGX Stain-Free™ Protein Gels) in 1X TGS buffer (Bio-Rad) and transferred using a semi-dry transfer (Bio-Rad Trans-Blot® Turbo Transfer System) to a 0.2μm nitrocellulose membranes (Bio-Rad). Blocking was done in a 5% skim milk in 1X TBST. Membranes were blotted using primary antibodies against the mouse α-FLAG antibody (Thermofisher, MA191878), rabbit α-actin (Thermofisher, MA5-32479), or rabbit α-GFP antibody (Thermofisher, A11122). Figures show representative immunoblots from at least three independent experiments for each set of transfected conditions.

Proteins were extracted from cell cultures with RIPA buffer (Sigma-Aldrich, Germany) along with proteinase and phosphatase inhibitors (Roche, Switzerland). Protein concentration was measured using a BCA kit according to the manufacturer’s protocol (BCA Protein Assay-Kit, ThermoScientific, Sweden). Protein extracts were then separated by SDS-PAGE using pre-cast gels (4–20%, Bio-Rad) in TGS buffer (Bio-Rad, Sweden). The proteins were transferred to nitrocellulose membranes (Bio-Rad, Sweden) using the TransBlot Turbo system from Bio-Rad. The membranes were subsequently blocked for 1 h with skim milk at 3% (w/v) in PBS, then washed 3 × 10 min in PBS supplemented with 0.1% (w/v) Tween 20 (PBS-T). Then, blots were incubated with primary antibodies in PBS-T, as mentioned above, overnight. Following this, we incubated the blots with secondary antibodies for 2 h. After the secondary antibody, we washed three times with PBS-T, and then the blots were developed using ECL Clarity (Bio-Rad) according to the manufacturer’s protocol and imaged using the ChemiBlot XRS+ system from Bio-Rad.

For SDS-PAGE, 14-µL samples mixed with 5.3 µL Laemmli buffer and 0.6 µL 2-mercaptoethanol were heated to 95 °C for 5 min. After cooling, 12-µL aliquots were transferred to the wells of TGX (Tris-Glycine eXtended) 4–20% gels (Bio-Rad Laboratories, Hercules, CA, USA) in an electrophoresis chamber filled with 1:10 diluted TGS buffer (Bio-Rad Laboratories, Hercules, CA, USA). Quantitative albumin standards of 500, 250, 125, 100, and 50 µg mL⁻¹ (Thermo Fisher Scientific, Dreieich, Germany) were run alongside. The gels were analyzed using the ChemiDoc MP imaging system and Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA). To determine the molecular weights of each band, Precision Plus Protein markers (Bio-Rad Laboratories, Hercules, CA, USA) were run in the first and last lanes.

Whole cell lysates were obtained with RIPA lysis buffer (Thermo Fisher Scientific) complemented with 1% halt protease inhibitor and 3% halt phosphatase inhibitor (Thermo Fisher Scientific) and mixed with Laemmli buffer. The mix was heated in a dry bath at 95°C for 7 minutes for further denaturation. Equal amounts of proteins were loaded and separated by SDS‐PAGE in TGS buffer (Bio‐Rad). Proteins were then transferred on a PVDF membrane (Bio‐Rad RTA transfer kit) on a semi‐dry transfer system (Bio‐Rad). Non‐specific binding sites on the membrane were blocked with TBS buffer with 0.1% tween and 5% milk. After an overnight incubation with primary antibodies in TTBS‐milk 1% (ACSS2 1:1000; Abcam ab66038, ß‐actin 1:1000; Cell Signaling Technology 4970S) and incubation with secondary antibody the day after, the protein bands were detected with SuperSignal West Pico Plus (Thermo Fisher Scientific).

A 125-µg protein sample obtained from fermented sheep milk was employed on a ready IPG strip (7 cm) from Bio-Rad, with a pH range of 3–10. Focusing via isoelectric means was conducted with electricity boundaries detailed by Panchal et al. (2020) (link). Following isoelectric focusing, the strip underwent successive immersion in buffer-I equilibrium (10 min) and equilibrium buffer-II (10 min). A 30-s rinse using Bio-Rad’s 1X TGS buffer was then performed before the strip was exposed to SDS-PAGE for two-dimensional (2D) analysis. The gel for the separating phase was prepared as per LaemmLi (1970) (link) and Carrasco-Castilla et al. (2012) (link).