Cells were seeded in tissue culture dishes as described above. For labelling, the cells were starved using IM medium (Glasgow minimal essential medium buffered with 10 mM Hepes, pH 7.4). After 1 h, the medium was replaced by IM with 3H-palmitate at 200 µCi/mL (American Radiolabeled Chemicals, US) for 2 h at 37 °C. Cell lysis, immunoprecipitation and SDS-PAGE were performed as above. The gels were fixed for 30 min with 10% acetic acid, 25% isopropanol in water and the signal was amplified for 30 min with NAMP100 (GE Healthcare, US). The gels were then dried and applied to an Amersham Hyperfilm MP (GE Healthcare, US). The radioactivity was visualized and quantify using a Typhoon TRIO (GE Healthcare, US).
Namp100
Manufactured by GE Healthcare
Sourced in United States
The NAMP100 is a laboratory equipment product designed to perform nucleic acid amplification testing (NAAT). It is a compact and automated instrument that can process multiple samples simultaneously, enabling efficient and reliable nucleic acid amplification analysis. The NAMP100 is capable of performing various NAAT techniques, such as real-time PCR, to detect and quantify specific genetic targets. This product is intended for use in clinical and research laboratory settings.
Automatically generated - may contain errors
6 protocols using namp100
1
Radioactive Fatty Acid Labeling
2
Pulse-Chase Analysis of MAPK Dynamics in Drosophila S2 Cells
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S2 cell culture, transfection, lysates, and western blotting were performed as previously described [59 (link)]. Pulse-chase radioactive labeling was performed by incubating S2 cells in ESF medium containing [35S]-methionine for 6 h. Cells were then harvested at different time points (0–36 h) following this incubation period, after which an α-HA immunoprecipitation was performed to enrich for HA-MAPK. Fluorographic reagent (NAMP100, GE Healthcare) was used to amplify [35S] signal. To visualize newly translated MAPK, we used a shorter labelling period of 4 h, after which cells were immediately lysed and submitted to immunoprecipitation. In both metabolic labeling experiments, RNAi treatment was performed for 5 d at a concentration of 15 μg/ml. Epoxomicin treatment was performed at a concentration of 1 μM (DMSO used for controls) 18 h prior to lysis of S2 cells. Whole fly lysates were prepared from 20 adults homogenized in 500 μl of RIPA buffer.
3
In Vitro Methylation Assay of ALDH1A1
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Methylation assay was performed in PBS (pH: 7.4) by incubating 2.5 μg of GST or 2.5 μg of GST-tagged ALDH1A1 or 1 μg of GST-GAR proteins with or without 6 μg of His-tagged catalytic domain of PRMT3 (His-PRMT3-CD) in the presence or absence of 0.76 μM radiolabeled S-adenosyl- L-[methyl-3H] methionine (SAM). The reaction mixture was incubated at 37 °C for 2 h and then separated in 12% SDS-PAGE. The gel was incubated with fluorographic reagent (GE Healthcare, NAMP100); dried and exposed to X-ray film for 14 days at −80 °C.
4
Metabolic Labeling of Viral Gag Protein
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HEK293T cells were transfected with vGag-RRE, Rev, empty vector or CCDC8. Two days post-transfection, the cells were washed with PBS, and starved for 30 minutes in methionine-cysteine free medium supplemented with 2% dialyzed fetal bovine serum (Invitrogen). Then, the cells were labeled for 20 min in medium containing 200 μCi [35S] methionine and cysteine (Express [35S] Protein Labelling Mix, NEG07200, Perkin Elmer Life Sciences). The pulse was ended by adding prewarmed DMEM supplemented with 10% fetal bovine serum and 5 mM of unlabeled methionine and cysteine. After various chase time periods, the cells were pelleted on ice and washed with ice cold PBS. For immunoprecipitation, the cells were lysed, and Gag was immunoprecipitated with anti-Gag antibody. The proteins were separated by 10% SDS-PAGE gel. The gel was fixed by buffer containing 40% ethanol, 10% acetic acid, 10% glycine at room temperature for 2 hours and amplified (NAMP 100, GE life Science) for 15 minutes, dried the gel and put the film on −80 °C freezer, then quantified by ImageJ.
5
SDS-PAGE Protein Visualization
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Proteins separated by SDS-PAGE were fixed in a solution of 25% (vol/vol) isopropanol, 10% (vol/vol) acetic acid, and 65% (vol/vol) double-distilled H2O (ddH2O). The radioisotope signal was amplified using a fluorometric solution (GE Healthcare; NAMP100), and gels were vacuum dried onto filter paper. The dried gels were exposed to X-ray film at −80°C prior to developing (62 ).
6
Radioactive Protein Labeling and Detection
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Cells were in tissue culture dishes as described above. For labelling, the cells were starved using IM medium (Glasgow minimal essential medium buffered with 10 mM Hepes, pH 7.4). After 1 h, the medium was replaced by IM with 3H-palmitate at 200 µCi/mL (American Radiolabeled Chemicals, US) for 2 h at 37°C. Cell lysis, immunoprecipitation and SDS-PAGE were performed as above. The gels were xed for 30 min with 10% acetic acid, 25% isopropanol in water and the signal was ampli ed for 30 min with NAMP100 (GE Healthcare, US). The gels were then dried and applied to an Amersham Hyper lm MP (GE Healthcare, US). The radioactivity was visualized and quantify using a Typhoon TRIO (GE Healthcare, US). 35
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