The RNA stability assay was performed using the Click-iT® Nascent RNA Capture Kit (Life Technologies). 3.5 h after DNA transfection, cells were treated with 10 μg/mL cycloheximide (Sigma) and 10 μM pactamycin (Sigma). At 4 h post transfection, the medium was replaced with DMEM containing 0.2 mM 5-ethynyl uridine (EU). 5 μg/mL actynomycin D (Sigma) was added 90 min post treatment with EU. Samples were collected 1.5, 2 and 4 h post EU incubation and total RNA was extracted with the RNeasy Plus Mini kit (Qiagen). Labelled RNAs were conjugated with an azide-modified biotin through a copper catalyzed click reaction. Streptavidin magnetic beads were used to capture labelled RNAs, which were then used as templates for reverse transcriptase-mediated cDNA synthesis for subsequent analysis using qPCR.
Pactamycin
Manufactured by Merck Group
Pactamycin is a broad-spectrum antibiotic produced by the bacterium Streptomyces pactum. It is a laboratory-grade compound used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Cycloheximide (chx), by Merck Group (2 mentions)
Click it nascent rna capture kit, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Pcmvr8, by Addgene (1 mentions)
Pcmv vsv g envelope plasmid, by Addgene (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Bortezomib, by Cambridge Bioscience (1 mentions)
Kribb11, by Merck Group (1 mentions)
Verrucarin a, by Merck Group (1 mentions)
3 protocols using pactamycin
1
Measuring mRNA Stability via Click-iT Assay
2
Synthesis and Compound Acquisition Protocol
3
Inhibitor Experiments and Ribosome Motility Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For inhibitor experiments, 500-µl cultures were incubated at 200 rpm in a 2-ml tube at 28°C with either benomyl at 30 µM (stock: 30 mM in DMSO; Fluka; Sigma-Aldrich), verrucarin A at 10 µg/ml (stock: 10 mg/ml in DMSO; Sigma-Aldrich), pactamycin at 100 µM (stock: 100 mM in DMSO; Sigma-Aldrich), cycloheximide at 100 µg/ml (stock: 100 mg/ml in DMSO; Sigma-Aldrich), or 100 µg/ml trichodermol (stock: 100 mg/ml in DMSO; provided by J. Zhao, Zhejiang University, China). For control experiments to analyze cytoplasmic GFP production driven under a crg promoter, cells were treated with verrucarin A, pactamycin, cycloheximide, or trichodermol for 3 h. For ribosome motility experiments, cells were cultured with benomyl for 30 min, verrucarin A for 1 h, or pactamycin for 10 min. Control cells were treated with the respective amount of solvent DMSO. Cells were placed onto a 2% agar cushion containing the respective inhibitor and immediately observed. For measurement of Rpl25-paGFP running time, cells were put onto a 2% agar cushion containing cycloheximide or trichodermol for 5 min and subsequently observed for 10 min. All experiments were done at least twice; the second experiment was usually nonquantitative.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!