Horseradish peroxidase conjugated anti rabbit igg

For protein analysis 50 mg of liver or intestine were homogenized in 700 μl of RIPA buffer containing 1% protein inhibitor cocktail (Santa Cruz, USA) and 1 μM PMSF, then centrifuged (14000 rpm at 4°C for 15 minutes). Protein concentration of the samples was measured in a Nano Drop spectrophotometer and 120 μg of protein was loaded on a 12.5% SDS-PAGE gel. After electrophoresis, proteins were transferred to a PVDF (Roche Applied Science) membrane. The Membrane was blocked with 3% skim milk in Tris-buffered saline containing Tween-20 (TBS-T) (Roche Applied Science) for 2 hrs at room temperature. The membrane was washed (three times, 15 min each) in TBS-T and then incubated with primary antibody for 1.5 hrs with anti rabbit polyclonal antibody LXR (1:300 dilution, Novus Biological (NB400-157)). After 3 washes in TBS-T, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:10,000 dilutions, Roche Applied Science) at room temperature for 1.5 hrs. The membranes were washed and exposed to ECL western blotting detection reagents (Roche Applied Science) and then exposed to films for 30s to 1 minute. Films were developed, scanned, and band densities were measured with Lab Work analyzing software (UVP, UK). Data are expressed as the percent ratio of the protein bands to β- Actin band [10 ,13 (link),16 (link)].

Crude homogenates suitable for immunoblot analysis were obtained by homogenizing one brain hemisphere in 15 mM KCl + 1 mM KH₂PO₄, pH 7.4, at 24,000 rpm/min for 90 sec in the cold, followed by centrifugation at 18,690 × g for 15 min at 4 °C. Proteins from homogenates stored at −80 °C (10 μg) were separated by 8% SDS–PAGE and transferred onto a nitrocellulose membrane (Bio-Rad, Hertfordshire, UK).
The milk-blocked membrane was then incubated overnight at 4 °C with the anti-CS (1:2000), or anti-OPA1 (1:2000), or anti-OMA1 (1:1000), or anti-DRP1 (1:1000) antibodies (Abcam, UK), subjected to a horseradish-peroxidase–conjugated anti-rabbit IgG (1:5000; Roche Diagnostics, Mannheim, Germany), and revealed with an enhanced chemiluminescence (ECL) Western blot analysis kit (Applied Biosystems) or by the alkaline phosphatase technique. ECL films were scanned densitometrically, and the optical density of bands was quantified using Image J version 1.38 software. Values obtained from the immunoblot quantification of CS were either used to obtain a measure of mitochondrial mass^{37 (link), 38} or to calculate the semi-quantitative measure of the different proteins relative to CS.