Ecl prime western blotting detection reagent kit

Cells were lysed in 4x Laemmli sample buffer and boiled for 5 min. Proteins were separated on 4–12% NuPAGE Bis-Tris Gel (Novex life technologies) and transferred on nitrocellulose membrane (0.45 µM). Membranes were blocked with 5% milk in PBS-1% Tween20 for 1 h and incubated overnight in primary antibodies. Membranes were then washed three times with PBS containing 0.05% tween and probed with respective secondary antibodies. Finally ECL Prime Western Blotting Detection Reagent kit (GE Healthcare) was used to develop the blots. Details of the antibodies used are provided in Supplementary Table 1.

For scFv-10D8 expression analysis, total protein extracts from E. coli cultures expressing scFv-10D8 were submitted to SDS-PAGE and transferred to PVDF membranes. The membrane was blocked PBS-T (Na₂HPO₄ 25 mM, NaH₂PO₄ 10 mM, pH 7.4, Tween 20 0.3%) plus 5% non-fat milk and incubated with anti-histidine primary antibody (1:3000) (Bio-Rad, USA) followed by horseradish peroxidase-conjugated anti-mouse secondary antibody (1:5000) (Bio-Rad, USA).
In order to analyse the recognition profile of mAb-10D8 and scFv-10D8, the total protein extracts of 2 × 10⁶ G strain epimastigote forms were electrophoresed (SDS-PAGE) and transferred to PVDF membranes. Membrane strip was incubated with mAb-10D8 (1:3000) followed by horseradish peroxidase-conjugated anti-mouse secondary antibody (HRP) (1:5000). Additional membrane strips were incubated with periplasmic (P) or cytoplasmic (C) fractions obtained from scFv-10D8 expression and also periplasmic fraction of unrelated scFv (scFv Loxo) followed by anti-His secondary antibody (1:3000) and finally the anti-mouse antibody conjugated to HRP (1:5000). Detection was performed with an ECL Prime Western Blotting detection reagent kit (GE Life Sciences). We used a scFv derived from LiMab7, an unrelated monoclonal antibody, as negative control, named here as scFv-Loxo [36 (link)].

Fully grown GV-stage oocytes from each group were isolated as mentioned earlier, and briefly washed with L-15 medium. Then, 30 oocytes per tube were collected in 10 µl of L-15 medium and snap-frozen in liquid nitrogen. Frozen oocytes were stored at −80°C before testing. Proteins were extracted using Bio-Rad sample buffer (161-0747) with 2-mercaptoethanol (Sigma-Aldrich; M7522) at 95°C for 10 min and then centrifuged at 16,000 g for 2 min; the supernatant was then collected. Proteins were separated using Bio-Rad 4-15% gradient gels (456-1084) and blotted on a PVDF membrane (GE Healthcare; 10061-494). Blots were blocked with 0.3% ECL Prime Blocking Reagent (GE Healthcare; RPN418) in Tris-buffered saline supplemented with 0.01% Tween (TBSTw) for 1 h. SMC3 (Abcam; ab9263; 1:2000) and γ-tubulin (Abcam; ab11316; 1:1000) primary antibodies were diluted in 0.3% blocking buffer at 4°C overnight. Blots were washed with TBSTw three times for 10 min each and then incubated with anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (GE Healthcare; NA931V and NA934V, respectively; 1:5000). Blots were washed with TBSTw three times each for 10 min and then developed with a ECL Prime Western Blotting Detection Reagent kit (GE Healthcare; RPN2232).

The volume of cell extracts corresponding to 20 μg of protein was subjected to SDS-PAGE electrophoresis. Proteins present in the gel were transferred to a PVDF membrane (GE Healthcare®). Membranes were blocked with 5% BSA solution in Tris-buffered saline (TBS) containing 0.1% Tween-20 (T-TBS), and incubated overnight at 4°C with anti-arginase-1 or anti-actin (1: 1,000). After 3 5-min washes with T-TBS, the membranes were incubated with peroxidase conjugated secondary antibodies (1: 10,000) for 1.5 h and incubated with chemiluminescent solution (ECL Prime Western Blotting Detection Reagent Kit-GE Healthcare). The immunoreactive bands were visualized in the ChemiDoc MP Imaging System (Bio-Rad®). Images were analyzed with ImageJ software (NIH/USA).

Total proteins were extracted from liver samples and prepared for Western blotting according to the manufacturer’s instructions (Minute Total Protein Extraction Kit; Invent Biotechnology, Inc., Plymouth, MN, USA). Protein concentrations were evaluated using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). The samples were size-fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polyvinylidene difluoride (PVDF) membranes were used to transfer the protein from gel to membrane. Afterwards, membranes were incubated with anti-phosphorylated PERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-α-tubulin (MBL Co., Nagoya, Japan) antibodies diluted in blocking buffer. Next, anti-rabbit IgG secondary antibody (GE Healthcare, Pittsburgh, PA, USA) was used for incubation of the membranes. An ECL Prime Western Blotting Detection Reagent Kit (GE Healthcare, USA) was utilized for visualization of the enhanced chemiluminescent (ECL) membranes and images were captured using an Image Quant LAS 500 (GE Healthcare, USA). The images were analyzed with the ImageJ software from the NIH.

To determine the amount of protein expression, a Western blot analysis was performed. After treatment, B16F10 melanoma cells were lysed with the RIPA lysis buffer (Biosesang, Seongnam, Korea). Whole cell lysates were separated by 9% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). After blocking with 5% skimmed milk in PBS containing 0.1% Tween 20, the membranes were probed with specific primary antibodies overnight at 4 °C and then further incubated with horseradish peroxidase-conjugated secondary antibodies. Bound antibodies were detected by chemiluminescence using an ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, Pittsburgh, PA, USA) following the manufacturer’s instructions. Antibodies for tyrosinase and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).