Peroxidase linked goat anti rabbit igg

Samples of the livers from each group were homogenized using ice-cold radio immunoprecipitation assay (RIPA) buffer and centrifuged at 12000 × g for 10 minutes at 4 °C. Thereafter, the total protein concentration was determined using a BCA protein assay kit (Abcam, ab102536). Samples (40 µg of protein per lane) were subjected to electrophoresis on a 10% SDS-PAGE gel and then transferred onto PVDF membranes (Millipore, ISEQ00010). After nonspecific blocking in 5% nonfat milk, the membranes were incubated with antibodies recognizing cytochrome C, Cox IV, Tomm20, ATGL and perilipin 5, overnight at 4 °C. After washing with TBST, the membranes were incubated with peroxidase-linked goat anti-rabbit IgG (1:5000, Bioworld Technology Inc., BS13278) for 2 h. Following incubation, the bound antibodies were visualized using High-sig ECL Western Blotting Substrate (Tannon). Immunoreactive bands were quantified using Quantity One software (Bio-Rad Laboratories) (Supplementary Figure S1).

Samples were homogenized in ice-cold with radioimmunoprecipitation assay (RIPA) buffer. The homogenates were subsequently centrifuged at 15,000 g for 10 min at 4°C. Then, the samples were subjected to electrophoresis in a 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred on to polyvinylidene fluoride (PVDF) membranes (Millipore, ISEQ00010). Nonspecific binding was blocked with 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature. Subsequently, the PVDF membranes were incubated at 4°C overnight with rabbit anti-LC3 (1:1000), anti-LC3A/B antibody (12741 Cell Signaling Technology, Danvers, Massachusetts, USA) and mouse anti-LAMP1 antibody (15665 Cell Signaling Technology, Danvers, Massachusetts, USA). Next, the membranes were washed with TBST and incubated with peroxidase-linked goat anti-rabbit IgG (1:5000, BS13278, Bioworld Technology Inc, Minneapolis, Minnesota, USA). Finally, the bound antibodies were visualized by using an the electrogenerated chemiluminescence (ECL) detection system (Vazyme Biotech, E411-04).

Five testis samples from different turtles in each month (February, May, July, October, and December) in 2014 were homogenized in ice-cold RIPA's buffer (25 mMTris/HCl (pH 7.6), 150 mMNaCl, 1% sodium deoxycholate, 1% Nonidet-P40, 0.1% SDS, 0.05 mM PMSF). The protein concentration was quantified by BCA protein assay (Thermo Fisher Scientific, Rockford, USA). Equal amount of each proteins samples (40 μg/lane) was subjected to electrophoresis on 10% SDS-PAGE and transferred to polyvinylidene di-fluoride (Millipore, Bedford, MA) membranes. After blocking in 5% fat-free dry milk, membranes were incubated with an LAMP-1 antibody (21997-1-AP, Proteintech Group, Chicago, IL) at a dilution of 1:1,000 for 12 h at 4°C. After washing, the membrane was incubated with peroxidase-linked goat anti-rabbit IgG (1:5,000, BS13278, Bioworld Technology Inc., Louis Park, MN) for 2 h. Bound antibodies were detected using the ECL detection system (Vazyme Biotech, China). Immunoreactive bands were quantified with Quantity One software (Bio-Rad Laboratories).