Labeling of mitochondrial translation products was performed using [35S]-methionine and [35S]-cysteine (EasyTag EXPRESS 35S Protein Labeling Mix, PerkinElmer) in intact cells for 1 h, as described previously [114 (link)]. Emetine (100 μg/ml) was used to inhibit cytoplasmic protein synthesis. Samples were loaded onto 15–20% polyacrylamide gradient gels and run at 10 mA for 16 h or until 1 h after the dye front ran out of gel [114 (link)]. Gels were fixed in a methanol-acetic acid solution, treated 30 min with Amplify (GE Healthcare), dried and exposed to Amersham Hyperfilm MP film with Hyperscreen intensifying screens (GE Healthcare) at -80°C for several days.
Amersham hyperfilm mp film
Manufactured by GE Healthcare
Amersham Hyperfilm MP film is a high-performance X-ray film designed for applications in molecular biology, biochemistry, and autoradiography. The film provides high sensitivity and contrast for the detection and visualization of radioactive signals.
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Lab products found in correlation
Easytag express35s protein labeling mix, by PerkinElmer (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Mini protean 3, by Bio-Rad (1 mentions)
Glycine, by GE Healthcare (1 mentions)
Tween 20, by Abcam (1 mentions)
3 protocols using amersham hyperfilm mp film
1
Labeling and Visualizing Mitochondrial Proteins
2
In vitro Kinase Assay for MYC2 Phosphorylation
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In vitro kinase assays were performed in a reaction mixture containing 40 mM Hepes, pH 7.5, 20 mM MgCl2, 2 mM DTT, 10 μCi [32P] γATP, 1× proteinase inhibitor cocktail, 1× phosphatase inhibitor cocktail, 4 μg MBP-MYC2 and 15 μg GST-OsGSK2 or GST. The mixture was incubated at 30°C for 0.5 h, then 7μl 5× SDS loading buffer were added to terminate the reaction. Thereafter, the reaction products were boiled at 100°C for 5 min, and then separated by 10% SDS-PAGE. The gel was stained with Coomassie blue and imaged, then exposed to GE Amersham hyperfilm MP film for 2 h to detect phosphorylation.
3
Western Blot Protein Detection
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Proteins were resolved on a discontinuous tris-tricine SDS-PAGE (5% stacking gel, 10% resolving gel) using Biorad Miniprotean III apparatus. Resolved proteins were blotted onto 0.22µm PVDF (Pierce) using a Novex semi-dry blotter and buffer containing 48mM Tris (Sigma), 39mM glycine (Sigma), 0.037% SDS (Sigma) and 20% Methanol (HPLC grade, Fisher).
Blotted membranes were blocked in 5% Marvel milk powder, 0.1% Tween20 (Sigma) in PBS (Oxoid) for one hour at room temperature followed by incubation with mouse anti-HA.11 (1:5000 , Covance, Clone 16B12) overnight at 4°C in heat-sealed bags. Blots were washed (5X5min) in PBS containing 0.1% Tween20 followed by incubation with HRP conjugated goat anti-mouse (1:10,000 in block, Abcam, ab6789) secondary antibody for 2h at room temperature in heat-sealed bags. Blots were washed (5X5 min) in PBS containing 0.1% Tween20. Blots were incubated with electro-chemiluminescent reagents (100mM glycine, 0.4mM luminal, 8mM 4-iodophenol and 10% dimethyl sulphoxide mixed 1:1 with 0.12% hydrogen peroxide) for one minute and exposed to Amersham Hyperfilm MP film (GE Healthcare).
Blotted membranes were blocked in 5% Marvel milk powder, 0.1% Tween20 (Sigma) in PBS (Oxoid) for one hour at room temperature followed by incubation with mouse anti-HA.11 (1:5000 , Covance, Clone 16B12) overnight at 4°C in heat-sealed bags. Blots were washed (5X5min) in PBS containing 0.1% Tween20 followed by incubation with HRP conjugated goat anti-mouse (1:10,000 in block, Abcam, ab6789) secondary antibody for 2h at room temperature in heat-sealed bags. Blots were washed (5X5 min) in PBS containing 0.1% Tween20. Blots were incubated with electro-chemiluminescent reagents (100mM glycine, 0.4mM luminal, 8mM 4-iodophenol and 10% dimethyl sulphoxide mixed 1:1 with 0.12% hydrogen peroxide) for one minute and exposed to Amersham Hyperfilm MP film (GE Healthcare).
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