Nupage 10 bis tris

Breast cancer cell lysates were prepared from CAP1 knockdown or control cells, and proteins were extracted using radioimmunoprecipitation assay buffer [RIPA; 10 mM Tris-HCl pH 7.4, 50 mM NaCl, 5 mM EDTA, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100 μM sodium orthovanadate, 1% Triton X-100] supplemented with protease and phosphatase inhibitors. Protein quantifications were performed using Pierce BCA Protein Assay Kit, according to the manufacturer’s instructions. Proteins were separated by pre-cast SDS-PAGE (NuPAGE 10% Bis-Tris, Invitrogen) and transferred to nitrocellulose membrane. The membrane was blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline with Tween-20 and incubated overnight at 4 °C with primary antibodies to CAP1 (Abcam; ab133655, 1:10000) or GAPDH (Merck; MAB374, 1:1000). The blots were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (CAP1, 1:2000; GAPDH, 1:10000) for 1 h and proteins visualized using SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific) and LI-COR Biosciences Odyssey Imaging System. Relative protein levels were quantified by densitometry using ImageJ software (NIH) and normalized against the GAPDH housekeeping protein.

Each protein extract (50 μg) was diluted in an appropriate volume of IPG rehydration buffer (7M urea, 2M thiourea, 66 mM DTT, 4% CHAPS, 0.5% ampholytes) and loaded on immobilized pH gradient strips (seven cm, linear pI gradient from 3 to 10) (Bio-Rad Italia, Hercules, CA, USA). The IPG strips were actively rehydrated for 6 h at 50 V and 20 °C, and isoelectrofocusing was carried out on a Protean IEF Cell (Bio-Rad, Hercules, CA, USA), starting with a voltage of 200 V for 1 h, then 1,000 V for 1 h and finally up to 4,000 V for a total of 25,000 Vh. The focused strips were incubated at RT in a reduction buffer (6M urea, 30% v/v glycerol, 2% w/v SDS, 50 mM Tris–HCl, pH 8.6, 2% w/v DTT) for 15 min and then in an alkylation buffer (6M urea, 30% v/v glycerol, 2% w/v SDS, 50 mM Tris–HCl, pH 8.6, 4.5% w/v iodoacetamide) for 15 min in the dark. The equilibrated strips were then embedded at the top of LDS precast homogeneous gels (NuPAGE 10% Bis–Tris, Invitrogen Corporation, Carlsbad, CA, USA) and electrophoretic separation was performed in an XCell SureLock Mini-Cell System (Invitrogen, Carlsbad, CA, USA) at RT, 200 V constant, 125 mA, 100 W for 45 min. The gels were stained with Colloidal Coomassie Blue (Candiano et al., 2004 (link)) and scanned with a ChemiDoc MP System densitometer (Bio-Rad, Hercules, CA, USA) at the resolution of 600 dpi.

Samples were resolved on SDS-PAGE 10% precast polyacrylamide mini gels (NuPAGE 10%, Bis-Tris, 1.0–1.5 mm; Invitrogen, Thermo Scientific). Untreated and treated samples were reduced by using NuPAGE Sample Reducing Agent (10×) (Invitrogen, Thermo Scientific), and Pierce LDS Sample Loading Buffer (4×) (Invitrogen, Thermo Scientific) was added to make proteins unfold in the presence of lithium dodecyl sulfate. Both reagents were diluted to 1× final concentration. Samples were heated up on a preheated thermomixer at 70°C/1100 rpm for 5 min and then pipetted into a 10% precast mini gel wells. A total of 1.5 µg of treated IFX and 4 µg of untreated IFX were loaded in wells. 20× Bolt MES SDS Running Buffer (Invitrogen, Thermo Scientific) was used at a final 1× concentration as running buffer, and 400 µl of Bolt Antioxidant (Invitrogen, Thermo Scientific) was added in to avoid sample reoxidation and maintain proteins in a reduced state during protein gel electrophoresis and protein transfer. The electrophoretic run was performed at a constant voltage of 200 V for 30 min. A ready-to-use Coomassie G-250 stain (SimplyBlue SafeStain; Invitrogen, Thermo Scientific) was used for 2 h at 37°C for visualizing protein bands on polyacrylamide gel.