The silencing efficiency was quantified by western blots using rabbit polyclonal anti-eIF2D antibody (12840–1-AP) from Proteintech and mouse polyclonal anti-GAPDH antibody (sc-1377179) from Santa Cruz Biotechnology. For that, 20 μg of protein extracts were loaded on 10% polyacrylamide SDS-PAGE. After migration, proteins were transferred to an Immobilin-P membrane (Millipore) at 10 V for 1 hr in a semi-dry apparatus (Trans-Blot SD) on a PVDF (polyvinylidene fluoride) membrane that had been previously activated with 100% methanol for few seconds and a transfer buffer pH 8 (25 mM Tris; 200 mM glycine; 20% ethanol). After transfer, the membrane was saturated for 2 hr by blocking buffer (5% milk, 0.05% Tween-20, PBS 1×). Primary antibodies were added at dilutions recommended by the manufacturers in blocking buffer, the membranes were incubated overnight at 4°C. Then, the membranes were washed three times by PBST (PBS 1×; 0.05% Tween-20) to remove the excess of primary antibodies. Then, membranes were incubated with secondary HRP-conjugated antibody for 1 hr at room temperature followed by three washing steps. The signal produced by reaction between HRP and ECL (Kit ECL Plus Western Blotting Detection System, GE Healthcare) was detected by chemiluminescence using imaging Chemidoc (Biorad).
Imaging chemidoc
Manufactured by Bio-Rad
The Imaging ChemiDoc is a versatile and powerful imaging system designed for a wide range of applications in molecular biology and biochemistry. It utilizes advanced CCD camera technology to capture high-quality images of chemiluminescent, fluorescent, and colorimetric samples. The system is capable of detecting a variety of detection methods, including gel electrophoresis, Western blotting, and protein and nucleic acid staining.
Automatically generated - may contain errors
2 protocols using imaging chemidoc
1
Quantification of Protein Silencing Efficiency
2
Quantification of Protein Silencing Efficiency
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The silencing efficiency was quantified by western blots using rabbit polyclonal anti-eIF2D antibody (12840-1-AP) from Proteintech, mouse monoclonal anti-eIF4E antibody (sc-9976) and mouse polyclonal anti-GAPDH antibody (sc-1377179) from Santa Cruz Biotechnology.
For that, 20 µg of protein extracts were loaded on 10% polyacrylamide SDS PAGE. After migration, proteins were transferred to an Immobilin-P membrane (Millipore®) at 10 Volts for 1 h in a semi-dry apparatus (Trans-Blot® SD) on a PVDF membrane (PolyVinyliDene Fluoride) that had been previously activated with 100% methanol for few seconds and a transfer buffer pH 8 (25 mM Tris; 200 mM glycine; 20% ethanol). After transfer, the membrane was saturated for 2h by blocking buffer (5% milk, 0.05% Tween-20, PBS 1X).
Primary antibodies were added at dilutions recommended by the manufacturers in blocking buffer, the membranes were incubated overnight at 4°C. Then, the membranes were washed three times by PBST (PBS 1X; 0.05% Tween-20) to remove the excess of primary antibodies.
Then, membranes were incubated with secondary HRP-conjugated antibody for 1h at room temperature followed by three washing steps. The signal produced by reaction between HRP and ECL (Kit ECL Plus Western Blotting Detection System, GE Healthcare®) was detected by chemiluminescence using imaging Chemidoc (Biorad®).
For that, 20 µg of protein extracts were loaded on 10% polyacrylamide SDS PAGE. After migration, proteins were transferred to an Immobilin-P membrane (Millipore®) at 10 Volts for 1 h in a semi-dry apparatus (Trans-Blot® SD) on a PVDF membrane (PolyVinyliDene Fluoride) that had been previously activated with 100% methanol for few seconds and a transfer buffer pH 8 (25 mM Tris; 200 mM glycine; 20% ethanol). After transfer, the membrane was saturated for 2h by blocking buffer (5% milk, 0.05% Tween-20, PBS 1X).
Primary antibodies were added at dilutions recommended by the manufacturers in blocking buffer, the membranes were incubated overnight at 4°C. Then, the membranes were washed three times by PBST (PBS 1X; 0.05% Tween-20) to remove the excess of primary antibodies.
Then, membranes were incubated with secondary HRP-conjugated antibody for 1h at room temperature followed by three washing steps. The signal produced by reaction between HRP and ECL (Kit ECL Plus Western Blotting Detection System, GE Healthcare®) was detected by chemiluminescence using imaging Chemidoc (Biorad®).
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