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LightShift Chemiluminescent RNA EMSA Kit


Capture and detect protein-RNA interactions on film without radioactivity.

LightShift Chemiluminescent RNA EMSA Kit

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:

  • 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

Product Details:

Protocol summary for performing RNA gel shifts with the LightShift RNA EMSA Kit.
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 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.

RNA gel shifts for iron-responsive protein (IRP) using 32P-labeled RNA probes and biotinylated RNA probes with the LightShift RNA EMSA Kit.
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.

Comparison of sensitivity between LightShift RNA EMSA Kit using biotinylated probes and traditional 32P-labeled RNA EMSA probes.
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.

RNA gel shift assay results for TERT, Lin28 and RNA Polymerase using the LightShift RNA EMSA Kit.
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.

RNA gel shift assay results for in vitro translated Aco1 using the LightShift RNA EMSA Kit. The Thermo Scientific LightShift Chemiluminescent RNA EMSA Kit is compatible with proteins expressed in the Human in vitro protein Expression System. RNA gel shifts were performed using in vitro translated Aco1 prepared with the Thermo Scientific Human In Vitro Protein Expression Kit (Part No. 88855). After in vitro, the translation reaction was buffer exchanged into 1X REMSA buffer using 0.5mL Thermo Scientific Zeba Spin Desalting Columns (Part No. 89882) and pre-cleared with 30µL of Thermo Scientific High Capacity Streptavidin Agarose beads (Part No. 20361) for 30 minutes at 4°C. The pre-cleared lysate was diluted 1:20 and 2µL was incubated with 5 nM Biotinylated IRE-RNA in 1X REMSA buffer supplemented containing 5% glycerol. 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. Lane 1. Reaction containing only the biotinylated IRE-RNA probe. Lane 2. Band shift of the IRE-RNA probe by addition of in vitro translated Aco1. Lane 3. Aco1 shift competed off by addition of 200-fold molar excess of un-labeled IRE-RNA. Lane 4. which was not competed by addition of 200-fold molar excess of a non-specific, unlabeled RNA.

References:

  1. Leibold, E.A. and Munro, H.M. (1988). Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5´ untranslated region of ferritin heavy- and light-subunit mRNAs. Proc. Natl. Acad. Sci. USA. 85:2171-5.
  2. McKinley, B.A. and Sukhodolets, M.V. (2007). Escherichia coli RNA polymerase-associated SWI/SNF protein RapA: evidence for RNA-directed binding and remodeling activity. Nucleic Acids Res. 35:7044-60.
  3. O’Connor, C.M., et al. (2005). Two purified domains of telomerase reverse transcriptase reconstitute sequence-specific interactions with RNA. J Biol Chem 280:17533-9.
  4. Piskounova, E., et al. (2008). Determinants of microRNA processing by the developmentally regulated RNA binding protein Lin28. J. Biol. Chem. 283:21310-4.
  5. Sukhodolets, M.V. and Jin, D.J. (1998). RapA, a novel RNA polymerase-associated protein, is a bacterial homology of SWI2/SNF2. J. Biol. Chem. 273:7018-23.

Related Resources:

Protein-nucleic acid interactions technical guide
Protein-RNA interaction detection technical guide
Gel shift assays (EMSA) technical guide

Related Products:

Pierce RNA 3' End Biotinylation Kit
Chemiluminescent Nucleic Acid Detection Module and Blocking Buffer
Biodyne B Nylon Membrane (positively charged for nucleic acid applications)
NE-PER Nuclear and Cytoplasmic Protein Extraction Reagent Kits


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
20158 LightShift Chemiluminescent RNA EMSA Kit
Formulation: Multi-component kit with reagents for RNA-protein binding reactions and blot detection
Sufficient For: 100 binding reactions and detection on 1000cm2 of membrane (10 mini-gel blots)

Kit Contents:
125nM Biotinylated IRE RNA Control 35μL
10µM Unlabeled IRE RNA Control, 25μL
2mg/mL Cytosolic Liver Extract, 50μL
10mg/mL tRNA, 100μL
10X REMSA Binding Buffer, 1mL
50% Glycerol, 500μL
2M KCl, 500μL
1M MgCl2, 500μL
DTT, lyophilized
Nuclease-Free Water, 1.5mL
5X REMSA Loading Buffer, 1mL
Stabilized Streptavidin-HRP, 1.5mL
Luminol/Enhancer Solution, 80mL
Stable Peroxide Solution, 80mL
Nucleic Acid Detection Blocking Buffer, 500mL
4X Wash Buffer, 500mL
Substrate Equilibration Buffer, 500mL
100-rxn kit Product Instructions for product #20158 LightShift Chemiluminescent RNA EMSA Kit MSDS for product #20158 LightShift Chemiluminescent RNA EMSA Kit $484.00

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20159 tRNA
Formulation: Transfer RNA at 10mg/mL in 10mM HEPES
Sufficient For: 100 binding reactions
100µL MSDS for product #20159 tRNA Certificate of Analysis for product #20159 tRNA $64.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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