LightShift Chemiluminescent RNA EMSA Kit

LightShift化学发光RNA EMSA 试剂盒特点：

•  安全：化学发光检测
• 灵敏：亚飞克级，等同于同位素灵敏度
• 快速、易用
• 可靠：试剂盒提供阳性对照，保证所有的检测系统正常工作

The Thermo Scientific LightShift Chemiluminescent RNA EMSA Kit provides a non-radioactive solution for studying RNA-protein interactions using an electrophoretic mobility-shift assay (EMSA). The RNA EMSA Kit uses biotinylated RNA probes and a chemiluminescent substrate system to achieve fast and safe detection of RNA-protein complexes with sensitivity equivalent to traditional ³²P-isotopic methods. The complete kit comes with all reagents required to set up and optimize protein-RNA binding conditions, a positive control for protein-RNA interactions and reagents for chemiluminescent detection of the nucleic acid interaction.

Highlights of the the LightShift Chemiluminescent RNA EMSA Kit:

• Sensitive – chemiluminescent detection comparable to radioactive detection
• Time-saving – develop X-ray films after 1- to 5-minute exposures, versus 16 hour exposures needed with radioactive systems
• Flexible – compatible with RNA probes biotinylated by different methods
• Easy to use – complete kit includes optimized reagents for binding reactions and RNA probe detection
• Non-radioactive – eliminate waste concerns from radioactive RNA probes

 

Protocol summary for the RNA electrophoretic mobility-shift assay using the Thermo Scientific LightShift Chemiluminescent RNA EMSA Kit.

 

About the LightShift Chemiluminescent RNA EMSA Kit

The LightShift Chemiluminescent RNA EMSA Kit is an in vitro technique for detection of protein-RNA interactions through changes in gel electrophoresis migration patterns similar to the popular DNA gel shift assay. In a RNA EMSA, a labeled RNA probe is incubated with a protein sample to initiate binding. Once a complex is formed, the sample is separated via non-denaturing polyacrylamide gel electrophoresis. Because RNA-protein complexes migrate more slowly than free RNA probes, the resulting difference in migration distance can be visualized with the RNA gel shift assay. Specificity of RNA-protein interactions are validated through binding competition with excess unlabeled RNA that decrease the signal of the specific interactions. Generally, mutated or irrelevant RNA probes are not expected to compete for specific interactions and should not reduce the intensity of specific band shifts when detected in the EMSA. The complete LightShift Chemiluminescent RNA EMSA Kit includes all reagents required to set up and optimize an RNA gel shift assay, including a positive control for RNA-protein complex formation.

The LightShift Chemiluminescent RNA EMSA Kit uses biotinylated RNA probes, streptavidin-HRP and chemiluminescent detection to provide sensitivity similar to using radioactive RNA probes and allow faster detection. Labeled RNA probes can be purchased commercially or generated through either run-off in vitro transcription reactions with biotinylated nucleotides or through enzymatic ligation of biotin tags to the 3' terminus of an RNA strand using the Thermo Scientific Pierce RNA 3' End Biotinylation Kit. The LightShift Chemiluminescent RNA EMSA Kit is effective for RNA probes biotinylated by any of these three methods; however RNA secondary structure may be affected by internal incorporation of biotinylated nucleotides during run-off in vitro transcription RNA probe synthesis. Therefore, for certain interactions, custom synthesized RNA probes or 3' end biotinylated probes may be required for proper protein-RNA interactions to occur.

 

About the LightShift Chemiluminescent RNA EMSA Kit Positive Control

The positive control included with the LightShift Chemiluminescent RNA EMSA Kit is the iron responsive element (IRE) RNA probe. IRE binding reactions are set up and detected in parallel with the other experimental samples. Under iron-starved conditions, the iron responsive protein (IRP) remains bound to the IRE RNA present in the cell, effectively suppressing translation of ferritin (an iron storage protein) and then transferrin iron receptor. Under iron-rich conditions, IRE binding activity is lost, and ferritin and transferrin are translated. This system is ubiquitous and yields a robust band shift. Incubating the positive control reaction with a 200-fold molar excess of unlabeled IRE RNA will reduce the specific IRE band-shift signal by approximately 70%, while a similar fold excess of an unrelated RNA probe will not significantly reduce the IRE band shift. These controls can be used in each RNA gel shift experiment to validate proper setup, electrophoresis, transfer and detection of the protein-RNA complex formation.

 

The Thermo Scientific LightShift Chemiluminescent RNA EMSA Kit is specific and provides results faster than radioactive methods. RNA gel shift assays were performed for the iron responsive element (IRE) using the LightShift Chemiluminescent RNA EMSA Kit. Binding reactions included 5nM (5.1fmol) of biotinylated IRE RNA protein 1X RNA EMSA Binding Buffer, 5% glycerol, 2µg of tRNA and 4µg of cytosolic liver extract containing iron-responsive protein (IRP). Reactions were incubated for 30 minutes at room temperature. Specificity was determined in replicate by adding 1µM unlabeled IRE RNA probe (specific control) or an unrelated unlabeled RNA probe (telomerase RNA). Reactions were resolved on a native 6% polyacrylamide gel in 0.5X TBE and transferred to a nylon membrane. Band shifts were detected using the Pierce Chemiluminescent Nucleic Acid Detection Module. A similar experiment was performed using 32P-end-labeled IRE RNA (5nM) using the conditions described above. Detection of the biotinylated IRE EMSA assay was completed after a 1 minute exposure to X-ray film. The 32P-labeled IRE EMSA was completed after a 16 hour exposure to X-ray film. Densitometry was performed on the scanned gels. In both assays, a robust IRE/IRP band shift was observed which was reduced by approximately 70% by addition of a 200-fold molar excess of unlabeled IRE RNA probe. However, addition of a 200-fold molar excess of unrelated RNA probe did not significantly reduce the band shift. These results demonstrate the robustness, sensitivity and specificity of the IRE/IRP positive control as well as the functionality of the LightShift Chemiluminescent RNA EMSA Kit.

 

Chemiluminescent detection with the Thermo Scientific LightShift RNA EMSA Kit has comparable sensitivity to radioactive detection. To demonstrate sensitivity of the LightShift Chemiluminescent RNA EMSA Kit, both biotin- and γ-32P-labeled RNA IRE probes were titrated from 0.25 to 1fmol and electrophoresed on a nondenaturing polyacrylamide gel. The biotinylated RNA probe was transferred to a positively charged nylon membrane (Part No. 77016), detected with the LightShift Chemiluminescent RNA EMSA Kit and exposed to film for 20 minutes. The radioactive RNA probe was exposed to film for 16 hours with an intensifying screen. Both systems were able to detect the IRE RNA probe at 50amol.

 

The Thermo Scientific LightShift Chemiluminescent RNA EMSA Kit accommodates different RNA and protein sources. Run-off transcription: Plasmid constructs were generated, and run-off transcription was performed using biotin-11-UTP or unlabeled UTP for hTR and Let-7. 3'-end-labeling: RNA for the RNA polymerase reaction was end-labeled using T4 RNA ligase and a modified biotinylated cytidine (Thermo Scientific Pierce RNA 3' End Biotinylation Kit, Part No. 20160). RNA probes were purified and incubated with lysates (TERT, Lin28) or purified RNA polymerase in 1X Binding Buffer. For Let-7/Lin-28, additional DTT and KCl were added, and the glycerol concentration was 2.5% for hTR/TERT. A 50- to 100-fold molar excess of unlabeled RNA was used for the competition reactions. A. hTR/TERT gel shift, B. Let-7/Lin28 gel shift, C. RNA/RNA polymerase gel shift.