Although there are some reports regarding different expression profiles of the housekeeping proteins in animal and experimental models ( 14, 15), housekeeping proteins are extensively used to confirm the quality of the experiments( 16, 17). However, to ensure that the result is not a false negative due to sample preparation errors or other experimental faults, it is necessary to have appropriate controls.
When a negative sample is encountered in western blot, it may be interpreted as negative. Western blot analysis is a widely used semi-quantitative method for specific determination of protein expression ( 13). In order to determine the minimum concen-tration of lysate proteins that could show a clear band in western blot with the antibody, a gradient of protein concentrations (0.5-20 μg) of Jurkat cell line lysate were used in the assay under non-reduced conditions. Negative controls were used to check the probable interaction with secondary antibody (HRP-conjugated sheep anti-rabbit antibodies), which included all the above-mentioned steps except for adding the anti-β-actin antibody to the membranes. After extensive washing with PBS-T, the filters were incubated with peroxidase-conjugated sheep anti-rabbit immunoglobulins (Avicenna Research Institute, Tehran, Iran) for 1 hr at room temperature followed by washing and developing with ECL chemiluminescent detection system (GE Healthcare, Uppsala, Sweden).
Filters were incubated with 10 ng/ml of anti-β-actin antibody for 1.5 hr at room temperature. All antibody incubations were performed in PBS-T containing 3% non-fat milk. The membranes were blocked overnight at 4☌ with 5% non-fat milk in PBS containing 0.05% Tween 20 (PBS-T). After electrophoresis, the resolved proteins were transferred onto Immobilon-PVDF membranes (Millipore Corporation, USA). Twenty μg of each sample was run on a 10% SDS-PAGE (100 V for 2 hr) under both reduced and non-reduced conditions. The supernatants were collected and protein concentrations in the lysates were measured by BCA Protein Assay Kit according to the manufacturer’s instructions (Thermo Scientific, IL, USA). The cell lysates were then centrifuged at 500× g for 30 min. Different samples were collected and each was lysed in 1 ml of lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 1% protease inhibitor cocktail (Sigma) for 1 hr on ice with 15 min intervals of vortexing for 30 sec. The ability of the antibody to recognize β-actin was assessed by western blotting.
Cell lysate preparation and western blot analysis
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