For analyses by western blotting, hOE-MSCs were collected by trypsination, counted and then centrifuged for 5 min at 300 g . Cell pellets were resuspended in 1× SDS loading buffer, heated at 95°C during 5 min, then each cell lysate was resolved by 8% SDS-PAGE and transferred onto a PVDF membrane (Thermo Scientific, Rockford, IL). After blocking membrane with 5% milk in PBS (Life Technologies, Gibco, Grand Island, NY) supplemented with 0.1% Tween-20 (Sigma-Aldrich, St. Louis, MO) (PBS-T), membrane was incubated at 4°C overnight with mouse monoclonal primary antibodies against IKAP diluted at 1:2000 (clone no 33, BD Biosciences, Franklin Lakes, NJ), anti-NOVA1 at 1:300 (cat. no WH0004857M7, Sigma-Aldrich, St. Louis, MO) or anti-β-actin at 1:10,000 (cat. no A2228, Sigma-Aldrich, St. Louis, MO) in 2.5% milk in PBS-T. Thereafter, membrane was then probed with mouse secondary antibodies diluted at 1:3500 (Sigma-Aldrich, St. Louis, MO) in 2.5% milk in PBS-T at room temperature for 45 min, and revealed by exposing pre-incubated membrane with ECL (ThermoScientific, Rockford, IL) using a G:BOX Chemi XT4 device (Syngene, Cambridge, UK). β-actin was used for normalization as the housekeeping protein.
G box chemi xt4 device
Manufactured by Syngene
The G:BOX Chemi XT4 device is a high-performance imaging system designed for gel and blot analysis. It features a sensitive CCD camera and a range of light sources to capture images of chemiluminescent, fluorescent, and colorimetric samples. The device is capable of producing high-quality images and data for various applications in research and diagnostics.
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Lab products found in correlation
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Tween 20, by Merck Group (2 mentions)
Mouse secondary antibodies, by Merck Group (2 mentions)
Anti β actin, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Ecl detection kit, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin primary antibody, by Merck Group (1 mentions)
2 protocols using g box chemi xt4 device
1
Western Blot Analysis of hOE-MSC Proteins
2
Western Blot Analysis of IKAP/hELP1
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Cell pellets were resuspended in 1X SDS loading buffer at 6x10 3 cells/µL, and then samples were heated at 95°C during 5 min. Thereafter, total protein from 10 µl of cell lysates were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Thermo Fisher Scientific, Rockford, IL). Membranes were blocked with 5% Milk in PBS supplemented with 0.1% Tween-20 (Sigma-Aldrich, St. Louis, MO) buffer for 1h at room temperature, and were then subsequently probed at 4°C overnight with mouse monoclonal anti-IKAP/hELP1 antibody diluted at 1:2000 (BD Biosciences, Franklin Lakes, NJ) in 2.5% Milk in PBST, followed by incubation with mouse secondary antibodies at 1:3500 (Sigma-Aldrich). As a control of equal protein loading, membranes were also probed with mouse anti-β-actin primary antibody used at 1:10000 (Sigma-Aldrich). Stained proteins were revealed by chemiluminescent detection using ECL detection kit (Thermo Fisher Scientific, Rockford, IL) on a G:BOX Chemi XT4 device (Syngene, Cambridge, UK).
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