Minigels protean tgx

SKOV3-SpyCatcher-BBζ cells were incubated with either 2000 nM myc-RFP-SpyTag or myc-RFP-SpyTag-DA for 1 h at 37 °C. Cells were lysed using RIPA lysis buffer with protease-inhibitor cocktail (Roche, cat. no. 5892970001) and centrifuged for 5 min. Lysate was then collected, and protein concentrations were quantified using a BCA assay (Thermo Scientific). Protein samples (80 μg) were mixed with loading buffer (Lammeli buffer; BioRad) containing 5% β-mercaptoethanol (BioRad) and incubated at 95 °C for 5 min. Samples were loaded in 4–15% Minigels protean TGX (BioRad) and run at 150 V for 1 h. A protein ladder (BioRad) was run along with the samples. Protein samples were transferred to a PVDF membrane (Millipore) at 100 V for 1 h. The membranes were washed with TBST (1% Tween; BioRad) and incubated with primary and secondary antibodies, including purified mouse anti-human CD3ζ (BD Pharmigen; 1:1000), anti-human/mouse/rat GAPDH, (R&D; 1:20 000), and peroxidase AffiniPure goat anti-mouse IgG (Jackson Immunology; 1:10 000). Membranes were washed three times in between the primary and secondary antibody incubation steps. Membranes were developed using the ECL prime Western blotting detection reagent (GE Healthcare no. RPN2236) and imaged using a GE ImageQuant LAS 4000 series imaging system.

Supernatant (30 μL) from constitutively expressed iToci (anti-hIL6Rα scFv-Fc-Myc tag) was mixed with loading buffer (Lammeli buffer; BioRad) with or without 5% β-mercaptoethanol (BioRad), boiled for 5 min at 95 °C and centrifuged at 14000g for 3 min. Samples were resolved on 4−15% Minigels protean TGX (BioRad) and run at 150 V for 1 h. A protein ladder (BioRad) was run along with the samples. Protein samples were transferred to a PVDF membrane (Millipore) at 100 V for 1 h. The membranes were washed with TBST (1% Tween; BioRad) and incubated overnight at 4 °C with Myc-Tag (9B11) Mouse mAb HRP Conjugate (1:1000) (Cell Signaling Technology, Cat# 2040S). Membranes were washed four times in between the primary and secondary antibody incubation steps. Membranes were developed using the ECL prime Western blotting detection reagent (GE Healthcare, RPN2236) and imaged (GE ImageQuant LAS 4000 series imaging system). Irrelevant constitutively expressed protein was used as a control.