LightShift™ Chemiluminescent RNA EMSA Kit

Catalog number: 20158

Thermo Scientific™  Related applications: Nucleic Acid Gel Electrophoresis & Blotting

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Description

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).

Features of 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

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.

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 but with 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.

Related Products
tRNA for LightShift™ Chemiluminescent RNA EMSA Kit
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Technique: Enhanced Chemiluminescence, Gel Shift, Membrane Blot
Assay Type: EMSA Assay
Product Size: 100 reactions
Detection Method: Chemiluminescent
For Use With (Equipment): X-Ray Film, myECL™ Imager

Contents & storage

Sufficient For: 100 binding reactions and detection on 1000 cm2 of membrane (10 mini-gel blots) • 125 nM Biotinylated IRE RNA Control 35 µL (store at -20°C)
• 10 µM Unlabeled IRE RNA Control, 25 µL (store at -20°C)
• 2 mg/mL Cytosolic Liver Extract, 50 µL (store at -20°C)
• 10 mg/mL tRNA, 100 µL (store at -20°C)
• 10X REMSA Binding Buffer, 1 mL (store at -20°C)
• 50% Glycerol, 500 µL (store at -20°C)
• 2M KCl, 500 µL (store at -20°C)
• 1M MgCl2, 500 µL (store at -20°C)
• DTT, lyophilized (store at -20°C)
• Nuclease-Free Water, 1.5 mL (store at -20°C)
• 5X REMSA Loading Buffer, 1 mL (store at -20°C)
• Stabilized Streptavidin-HRP, 1.5 mL (store at 4°C)
• Luminol/Enhancer Solution, 80 mL (store at 4°C)
• Stable Peroxide Solution, 80 mL (store at 4°C)
• Nucleic Acid Detection Blocking Buffer, 500 mL (store at 4°C)
• 4X Wash Buffer, 500 mL (store at 4°C)
• Substrate Equilibration Buffer, 500 mL (store at 4°C)

Documents

Manuals & protocols