Spectra multicolor broad range protein ladder

For the SDS-PAGE analysis in both reducing and non-reducing conditions, 50 µL milk was mixed with 950 µL buffer (50 mM Tris buffer containing 1% SDS, pH 8.0). Then, 65 µL of the diluted sample was mixed with 25 µL NuPAGE LDS sample buffer and either 10 µL 1 M DTT (Invitrogen, Naerum, Denmark) for reducing and Milli-Q water for non-reducing conditions. Proteins were separated using a 4–12% NuPAGE Novex Bis-tris gel using 5 µL of the prepared samples. Spectra™ Multicolor Broad range protein ladder (ThermoFisher Scientific, Waltham, MA, USA) was also loaded to the gel (5 µL). Electrophoretic separation was carried out applying a voltage of 200 V in cassettes containing cold MOPS SDS running buffer (Invitrogen, Naerum, Denmark). Afterward, staining of the gel was done using Coomassie Blue (100 mL equilibrium buffer and 1 mL brilliant blue) on a rocking shaker (Buch & Holm A/S, Herlev, Denmark) overnight. The gels were transferred to Milli-Q water for destaining. The gel was scanned using an Epson Perfection V750 pro scanner (Epson, Nagano, Japan). Semi-quantification of the identified bands was performed using image analysis software (TotalLab 100, Nonlinear Dynamics, Durham, NC, USA).

SDS-PAGE electrophoresis was performed on hydrolysate samples using a 12% Precise™ Protein Gel on a Hoefer™ Mighty Small™ II Mini Vertical Electrophoresis System. Hydrolysate samples and sample buffer were mixed in a 1:1 ratio and boiled for 5 min. Afterwards, 20 μL of the reduced protein sample was used to load on to the separating gel. The separation was performed under 40 mA current for 100 min. Spectra Multicolor Broad Range Protein Ladder (Thermo Scientific), a protein standard containing 10 pre-stained proteins with molecular weights ranging 10–260 kDa, was used. The gel was stained with Coomassie Brilliant Blue R-250. Molecular weights (Mw) were then estimated on the basis of the protein standard.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (1970) [63 (link)]. The protein molecular weights were calibrated using the Thermo Scientific™ Spectra™ Multicolor Broad Range Protein Ladder (10−260 kDa). Trimeresurus nebularis venom (10 µg) was loaded onto a 15% gel and the electrophoresis was performed under reducing conditions at 80 V for 2.5 h. Proteins were stained with Coomassie Brilliant Blue R-250 for visualization.

Tumor specimens were sliced to small pieces and homogenized in 200 µL RIPA buffer (with protease inhibitor cocktail), insoluble materials were removed by centrifugation at 12,000 g for 10 min at 4 °C. After measuring total protein concentration by BCA method, 15 µg of individual sample of 4 groups were loaded into 4 12% SDS-PAGE gel with Spectra™ Multicolor Broad Range Protein Ladder (Thermo Fisher) and then run in Mini-PROTEAN® Tetra Cell Systems (Bio-rad). Proteins were then transferred to Immun-Blot PVDF Membrane(Bio-rad) by Trans-Blot® Turbo™ Transfer System (Bio-rad) in 25 V, 1.3 mA (constant current) for 5 min. The membrane was blocked in 5% fat-free milk in room temperature for 2 hours and then incubated with anti-survivin (Rb mAb, 1:5000 diluted in 2% milk, Abcem) and anti-GAPDH (Rb mAb, 1:1000 diluted in 2% milk, ProSci) in 4 °C for overnight. TBST was used to wash the membrane for 10 min × 3 times and then the membrane was incubated with secondary antibody (Goat pAb to Rb IgG, 1:20000 diluted in 2% milk, Abcem) at room temperature for 40 min then the TBST wash step was repeated. 4 membranes were then incubated together with ECL substrate (Bio-rad) for 3 mins. Membranes were then exposed to Amersham Hyperfilm^TM (GE Healthcare) together and process by Series 2000A Processor film developer (TiBA). Film were scanned (Epson V550) and quantify by ImageJ 2.0.

Transfected cells were lysed by sonication. 150 μg and 10 μg of denaturated protein extracts were separated on 12% and 15% SDS-PAGE (SDS-polyacrylamide gel electrophoresis) for tenascin-C and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) detection respectively. Spectra Multicolor Broad Range Protein Ladder (Thermo Fisher Scientific) was used as the size marker. Subsequently the wet transfer onto PVDF membrane with 0.45 μm pore size (GE Healthcare) was performed in transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol). The membranes were placed in the SNAP i.d. 2.0 apparatus (EMD Milipore), where it was blocked with a 0.5% solution of skimmed milk in PBS. For detection of tenascin-C the TNC polyclonal antibody (H-300, Santa Cruz) was used, while GAPDH was detected using monoclonal antibody (0411, Santa Cruz). Antibodies were diluted 1:500 in 3% BSA (Sigma-Aldrich). Membranes were incubated with the primary antibody for 10 min or overnight,. Afterwards, secondary anti-rabbit IgG and anti-mouse IgG antibodies conjugated with horseradish peroxidase (HRP) (Sigma-Aldrich) were used. Detection of a protein was carried out with WesternBright Sirius Chemiluminescent Detection Kit (Advansta). Intensity of individual bands was analyzed qualitatively by Multi Gauge ver. 2.0 (Fujifilm).

20 μg of total protein per sample with 2 × SDS loading buffer was loaded onto pre-cast 4%–12% Bis-Tris gels (Life Technologies) alongside Spectra Multicolor Broad range Protein ladder (Thermo Fisher). Samples were separated by electrophoresis. Protein was transferred to PVDF membrane. Membranes were incubated with blocking solution (5% (w/v) non-fat milk/PBS), and incubated with primary antibody overnight at 4°C. After washing, membranes were incubated with secondary antibody for 1 h at room temperature and exposed using 680 nm and/or 780 nm laser (LiCor Odyssey, Ferrante, Giorgio et al.), or incubated with SuperSignal West Femto reagent (Pierce) and exposed using Chemiluminescence settings on ChemiDoc MP imaging system (Bio-Rad)

Ten μg per well of purified recombinant proteins were loaded onto an SDS-12% polyacrylamide gel (Life Science, Hercules, CA, USA). Gels were either stained with Coomassie Brilliant Blue or used for Western blot analysis. For Western blot analysis, the gel was transferred to a nitrocellulose membrane which was then blocked with 5% BSA (Sigma-Aldrich) for 2 h at RT, washed four times with Tris-buffered saline (TBS; 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.5% Tween 20) and incubated with pooled sera collected from vaccinated cattle at day 60. Sera with primary antibodies were used at a 1:300 dilution in TBS, and the membrane was incubated overnight at 4 °C and washed four times with TBS. The membrane was then incubated with an anti-bovine IgG-horseradish peroxidase (HRP) conjugate (Sigma-Aldrich) diluted 1:5000 in TBS with 3% BSA (BSA/TBS). The membrane was washed five times with TBS and finally developed with 3,3′, 5,5′-tetramethylbenzidine (TMB) stabilized substrate for HRP (Promega, Madrid, Spain) according to the manufacturer recommendations. Molecular weight markers (Spectra multicolor broad range protein ladder; Thermo Scientific) were used.