LightShift™ Chemiluminescent RNA EMSA Kit - FAQs

NativePAGE凝胶已被成功用于EMSA，对2种纯化蛋白质间的相互作用进行分析。但是，我们尚未测试将NativePAGE凝胶用于EMSA以分析核酸（DNA或RNA）与蛋白质或蛋白质复合物间的相互作用。

EMSAs (also called gel shifts, band shifts, gel retardation assays, or mobility assays) have been used extensively for studying protein-DNA interactions. Because protein-DNA complexes migrate more slowly through a native polyacrylamide or agarose gel than DNA alone, individual protein-DNA complexes can be visualized as discrete bands within the gel using chemiluminescence or radioisotopic detection.

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You can identify the presence of a specific protein in a shifted band using a supershift assay. An antibody that is specific for the protein of interest is added to the binding reaction. Binding of the antibody to the protein causing the gel shift will often result in decrease in the mobility of the shifted complex, although other effects are possible (such as the disappearance of the shifted band). The order of addition of the components for a supershift is often critical; generally the antibody should be added last. We suggest this order:
- Water, buffer, any additives
- Poly dI-dC
- Nuclear extract (allows for non-specific proteins to bind to the poly dI-dC competitor)
- Biotinylated-DNA duplex
- Antibody (we use about 1 µg)
Incubate for 20 min.

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We recommend running run a cold probe competition lane (cold probe + label probe ++ nuclear extract) to see which band becomes fainter when compared to the label probe (label probe + nuclear extract) lane. With the cold probe competition lane, you can better distinguish which is the shifted band of interest.

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We recommend using the Chemiluminescent Nucleic Acid Detection Module Kit, Cat. No. 89880 as it has been optimized with the control probes in these kits. Results with other HRP-compatible substrates may vary depending on the quality or characteristic of the chemiluminescent substrate used.

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Alkaline transfer contains NaOH and is typically used for northern and Southern blots. We do not recommend alkaline transfer for EMSA. We recommend the use of 0.5X TBE buffer for transfer of EMSA binding reactions. TBE transfer is near-neutral pH condition.

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We recommend using positively-charged nylon membranes for transfer of EMSA reactions. Nitrocellulose membranes and PVDF membranes are not ideal as they strongly bind to protein but only weakly bind to DNA.

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We recommend using a native 4-6% polyacrylamide gel in 0.5X TBE buffer. The appropriate polyacrylamide percent depends on the size of the target DNA/RNA and the binding protein. Our precast 6% DNA Retardation gels may be used.

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The control extract provided is E. coli expressing recombinant EBNA, and the control probe is the Biotin-EBNA Control DNA. These controls should be run together. You can use the control extract, control probe, and cold control probe to affirm the binding reaction, running of the gel, transfer, and detection steps of the protocol.

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You can generate the EMSA probe using PCR with 5'-biotin modification. Avoid using probes that are internally labeled with biotin as this may interfere with the binding of target proteins.

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Many different methods can be used to anneal complementary oligo pairs. Please refer to our Tech Tip - Anneal complementary pairs of oligonucleotides: https://tools.thermofisher.com/content/sfs/brochures/TR0045-Anneal-oligos.pdf

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The EMSA probes should be diluted in dH2O and stored at -20 degrees C.

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Here are our recommendations:
- Typically, end-labeled, double-stranded DNA probes of 20-35 bp length are used, but other sizes are possible (note that the EBNA control probe in the kit is 60 bp long).
- Probes can be ordered with a 5'-biotin modification. They can be also enzymatically labeled on the 3' end using the Pierce Biotin 3' End DNA Labeling Kit (Cat. No. 89818).
- Single-stranded labeled probes should be annealed.
- Probes shorter than 20 bp may not bind the target protein well; longer probes can work but may require additional optimization or adjustments to gel running conditions.
- It is critical to have positive and negative controls in the EMSA assay.

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Yes. You may use the same method that was used with the G2 Fast Blotter: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/protein-biology-application-notes/transfer-emsa-gels-using-g2-fast-blotter.html

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Here are some suggestions to improve the results:

• Make sure that the target RNA used is end-labeled with biotin. Optimize labeling method and test for biotin labeling efficiency before assay. 
• Increase target RNA concentration.
• Check integrity of target RNA to make sure it is not degraded.
• Check the transfer protocol to make sure that the transfer is efficient.
• Use the right type of membrane (Biodyne B Nylon Membrane, Cat. No. 88518).
• Cover the membrane completely during incubations to make sure it doesn’t dry out during the detection steps.
• Make sure that the crosslinking is efficient.
• Make sure that the 4X wash buffer is diluted to 1X.
• Increase film exposure time during detection.

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Use a nonspecific competitor RNA such as tRNA or heparin.

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Speckling/spots can be caused by precipitate in the HRP conjugate or by air bubbles. Precipitate in the HRP conjugate can be removed by filtering the conjugate through a 0.2 µm filter or by centrifugation for 1 minute at maximum speed while air bubbles between the gel and the membrane should be removed before transfer.

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Here are possible causes and solutions:

- Particulate in Blocking Buffer or Wash Buffer: Gently warm until no particulate remains.
- Excess free biotin in biotinylated RNA preparation: Remove excess biotin by extracting with chloroform or using a Sephadex Column.
- Excess biotin in extract preparation: Remove endogenous biotin using High-Capacity Streptavidin Agarose (Cat. No. 20357) to pre-clear extract.
- Contaminants in the TBE: Use high-quality reagents or filter TBE through a 0.2 µm filter before use.
- The transfer unit or sponges used were dirty: Use clean equipment and sponges that were not previously used for western blotting.

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Here are possible causes and solutions:

- RNA/protein complex may have been disrupted by vortex mixing or heating: Try running the gel with cold buffer.
- Not enough extract was used: Use more extract.
- Extract degraded: Add protease inhibitors to the extract.
- System not optimized: System optimization can be achieved by using additives such as KCl, glycerol, MgCl2 and/or detergents and determining their effects on the shift. Comparing the methods used in journal articles that successfully performed EMSA experiments with the proteins you are testing may help with optimization.

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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 RNA 3' End Biotinylation Kit (Cat. No. 20160). 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.

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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 a DNA gel shift assay. In an 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. 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.

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NativePAGE gels have been successfully used to perform EMSA showing protein-protein interactions between two purified proteins, but we have not tested NativePAGE gels for EMSA analysis of interactions between nucleic acids (either DNA or RNA) and a protein or protein complex.

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