Mini protean tgx stain free

PRL full length protein sequences fused to an N-terminal 6xHis-tag was cloned into pET28b bacterial expression vector. Proteins were expressed in One Shot BL21 Star bacteria (Invitrogen, Cat # C601003) by induction with 0.5 mM IPTG (Fisher Scientific, Cat # BP175510) for 16 h. Cells were resuspended in 10 ml of lysis buffer [300 mM NaCl (VWR Cat. No. BDH9286), 20 mM Tris pH 7.5, 10 mM Imidazole pH 8.0 (Sigma-Aldrich I2399), 1:1000 protease inhibitor cocktail (Sigma-Aldrich Cat. No. P8465)] per gram of cell pellet and lysed using a microfluidizer (Avestin, EmulsiFlex-C5). Protein was isolated using Ni–NTA Resin (VWR, Cat # 786–940) and eluted with 2 mL of elution buffer (300 mM NaCl, 20 mM Tris pH 7.5, and 250 mM Imidazole pH 8.0). Following cleavage with TEV protease, samples were reapplied to Ni–NTA column to remove uncleaved protein as well as TEV. Samples were further purified using an S200 column on a Superdex 10/300 in buffer containing 100 mM NaCl and 200 mM HEPES pH 7.5. Purified fractions were then run on 4–20% Mini-PROTEAN TGX Stain-Free (Bio-Rad). The purest fractions were pooled, concentrated together, flash frozen and stored at -80 °C.

To analyse actin depolymerisation, we first incubated 2 µM G-actin with 2 µM Phalloidin in AP buffer for 1 h at RT. Then, 0.5 to 1 µM of Mc modules was added and incubated for 1 h at RT. G-actin was diluted from 2 to 0.1 µM in AP buffer (alone or with 0.5 or 1 µM of Mc modules) for 20 min, centrifuged at 100,000×g and pellets were separated on SDS-PAGE and detected by Stain-Free technology in the gel (Mini-PROTEAN® TGX Stain-Free™, Biorad). Protein bands were quantified from triplicate blots of independent experiments using ImageJ software (National Institutes of Health, Bethesda, MD). The remaining actin filaments were calculated as a percentage of the input. Kinetic depolymerisation of 2 µM of 40% pyrene-labelled actin, alone or in the presence of 0.5 or 1 µM of Mc modules, was monitored after dilution to 0.1 µM actin in AP buffer (alone or with 0.5 or 1 µM of Mc modules).

Specific EV protein markers and selected candidates were assessed in EVs obtained from PFP samples (n = 3). A total of 12.5–20 μL of isolated EVs were lysed using thermal shock (3× switch from 37 °C to liquid N2). The obtained proteins were loaded in SDS-PAGE gels (4–20% Mini-PROTEAN TGX Stain-Free, Bio-Rad, Hercules, CA, USA) and transferred onto nitrocellulose membranes (Trans-Blot Turbo, Bio-Rad). The blots underwent an overnight incubation with primary antibodies, including 0.125 μg/mL mouse anti-AIP1/Alix (clone 49/AIP1, #611620, BD Bioscience, Mississauga, ON, Canada), 1 μg/mL mouse monoclonal antihuman EMMPRIN/CD147 (clone IT10C5, #21451471, Immunotools, Friesoythe, Germany), 1 μg/mL goat polyclonal antihuman apolipoprotein B100 (AF3260, Novus Biologicals, Centennial, CO, USA), 5 µg/mL antihuman NECTIN-2 (MA5-34802, Invitrogen, Carlsbad, CA, USA), 2 µg/mL antihuman PECAM-1 (M0823, Dako, Carpinteria, CA, USA ), and 1:500 dilution antihuman IGFBP-2 (MA5-15400, Invitrogen). Peroxidase-conjugated secondary antibodies (1:5000 and 1:10,000 dilution) were applied as needed. Peroxidase activity was detected with a chemiluminescent substrate (TMA-6, Lumigen, Southfield, MI, USA), and images were acquired with the Chemidoc imaging system (Bio-Rad).

Proteins were extracted from cell pellets using lysis buffer (1 M Tris HCl pH 8, Triton X-100 1%, and Na deoxycholate 0.25%) with the addition of PMSF 1 mM. The proteins were then quantified colorimetrically using the BioRad protein Assay Reagent (Bio-Rad). Absorbance was measured at 595 nm with ChroMate microplate reader (Awareness Technology). The proteins were separated on polyacrylamide gels SDS-PAGE (10%, gel precast Mini-PROTEAN® TGX Stain-Free™, Bio-Rad) and transferred to 0.2 μm nitrocellulose membranes by electro blotting using the Trans-Blot Turbo Blotting System (Bio-Rad). The resulting blots were blocked with 5% nonfat dry milk solution in TBST. Anti-GAPDH (Cell Signaling) (1 : 20000), anti-E2F6 (Santa Cruz Biotechnology) (1 : 500), and PARP-1 (Santa Cruz Biotechnology) (1 : 500) primary antibodies were used. Incubation was performed overnight at 4°C, and bands were revealed after incubation with the recommended secondary antibody coupled to peroxidase using ECL (GE Healthcare). Scanned images were quantified using ImageJ software and normalized to GAPDH.

Aliquots of 10 to 20 μg of total proteins were prepared and electrophoresed from AV extracts on SDS polyacrylamide gels (4–15% polyacrylamide, Mini-PROTEANTGX Stain-Free, BioRad) and transferred to Hybond-C Extra nitrocellulose membranes (BioRad). Membranes were incubated with primary antibodies for CD36 (Santa Cruz Biotechnology) and β-actin (Sigma-Aldrich); and with secondary antibodies for mice and rabbits (GE Healthcare). Blot densitometry analyses were performed using Image Lab software. β-actin and stain free were used as loading controls for normalization and the net band densitometry was expressed as fold changes of arbitrary units (AU). Positive blots were detected with a chemiluminescence method (ECL, Amersham Biosciences) and images acquired with the Chemidoc MP Imaging system (Bio-Rad). All western blots were performed at least in triplicate for each experimental condition. Semiquantitative analyses were performed by band densitometry using the Image Lab software (Bio-Rad).

Cells were collected in Triton X-100 RIPA buffer and sonicated. After a denaturing step of 20 min at 95 °C in Laemmli buffer, 10 µg of proteins were resolved in 4–15% polyacrylamide gels (Mini-PROTEAN TGX Stain-Free, Bio-Rad) and transferred to a nitrocellulose membrane (Proten 0.2 µm NC, Amersham). Membranes were blocked in TBST containing 5% (wt/vol) non-fat dry milk for 30 min at RT and incubated with the primary antibody overnight at 4 °C. The day after, membranes were washed in TBST, incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at RT, and washed again. Proteins were detected with the chemiluminescence detection reagent Clarity Max Western ECL Substrate (Bio-Rad) and visualized using the ChemiDoc MP Imaging System (Bio-Rad).
Primary antibodies (diluted to between 1:2000 and 1:5000 in TBST containing 5% milk) were rabbit Myc-Tag (71D10, Cell Signaling Technology), rabbit β-Actin (#4967, Cell Signaling Technology), mouse mCherry (Living Colors, Clontech), and rabbit Pumilio 1 (C2C3, C-term, GENETEX). Horseradish peroxidase-linked secondary antibodies (diluted to 1:5000 in TBST containing 5% milk) were anti-rabbit IgG (#7074, Cell Signaling Technology) and anti-Mouse IgG (H + L) (W4021, Promega).