Protein-Nucleic Acid Interactions Support—Getting Started

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

Biotin end-labeled DNA containing a putative or known binding site is incubated with a nuclear extract or purified factor. This reaction is then subjected to gel electrophoresis on a native polyacrylamide gel and transferred to a nylon membrane. Because DNA-protein complexes migrate slower than DNA alone in a native gel, a “shift” in the migration of the labeled DNA occurs. The biotin end-labeled DNA is detected using a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate developed for the LightShift™ Kit. The signal is then detected with X-ray film or a high quality CCD camera.

DNA labeled on the 5'- or 3'-end can be ordered directly from your oligo supplier or prepared using a biotin end-labeling kit such as the Biotin 3' End-Labeling Kit (Cat. No. 89818). Internal biotin labels may inhibit binding of the DNA binding protein and are not recommended.

The binding conditions for each experiment must be optimized empirically for the proteins tested. Journal articles are an excellent source for information on how to set up EMSA binding reactions. If an article describes an EMSA for your protein of interest but used ³²P labeled probes, repeat the binding conditions with biotinylated probe in place of the ³²P labeled probe. Although reaction components are provided with the LightShift™ Chemiluminescent EMSA Kit, these reagents may not be suitable for individual applications and may be adjusted as needed.

The amount of protein extract needed for a binding reaction depends on how much active DNA binding protein is in the sample. The LightShift™ Kit is sensitive and will easily detect 5 fmol of active protein bound to 5 fmol of biotinylated probe. If the protein being studied is abundant, 0.25 μg of a cell lysate may be sufficient for each binding reaction. However, if the protein of interest is rare, 10 μg or more of cell lysate may be needed. Using a large excess of protein extract may lead to high background signal and non-specific bands.

As a control, unlabeled DNA of the same sequence as the biotin-labeled DNA is added in excess to compete with the biotin-labeled DNA. This results in a decrease or elimination of the “shift”, verifying that the protein-DNA interaction was sequence specific.

This has not been tested but may be possible. A better alternative is to perform a DNA binding protein pull-down assay using a probe. The following journal article is a good example of how the LightShift™ Chemiluminescent EMSA Kit and pull-down assays were used to detect a transcription factor bound to a DNA probe: Ragione, A.L., et al. (2003), J. Biol. Chem. 278(26):23360-8.

Positively charged nylon membrane must be used (e.g., Biodyne™ B Nylon Membrane, Cat. No. 77016).

The magnitude of mobility shift will vary in different systems and depends upon the abundance and activity of the target protein. Using the control provided with the kit, approximately 50% of the biotin-labeled DNA should be shifted.

Multiple shifts in an EMSA are most likely caused by the DNA binding to multiple forms of the protein such as a monomer vs. a dimer, or perhaps from multiple factors binding to the same site or to multiple binding sites within the target DNA (especially common with longer duplexes).

A supershift assay is a method for positively identifying a protein:DNA interaction on an EMSA antibody (typically 1 μg) is added to the binding reaction. During electrophoresis, the antibody:protein:DNA complex migrates slowly, causing a “supershift” compared to the “shift” caused by a protein:DNA complex. Not all antibodies will cause a supershift. Some antibodies do not bind to proteins once they are bound to DNA. Some antibodies can prevent protein:DNA interactions but can still be used to confirm the identity of a protein that causes a shift in the absence of the antibody.

Yes, the LightShift™ Chemiluminescent EMSA Kit can be used to detect supershifts. However, not all antibodies will work for supershift assays. Some antibodies will prevent protein:DNA interactions. In addition, the order in which the components of the binding reaction are assembled may affect the results of a supershift assay. Generally, 1 μg antibody is added as the last component in the binding reaction. For examples of how the LightShift™ Chemiluminescent EMSA Kit was used to detect supershifts, see the following references:

Adimoolam, S. and Ford, J.M. (2002). PNAS. 99(20):12985-90
Magid, R., et al. (2003). J. Biol. Chem. 278(35):32994-9
Ragione, F.D., et al. (2003). J. Biol. Chem. 278(26):23360-8
Rinaldi, A.L., et al. (2002). Cancer Research 62(19):5451-6

EMSAs may be performed with either polyacrylamide or agarose gels depending upon the resolution requirements of the study system. Traditionally, 4-6% non-denaturing polyacrylamide gels are used. If agarose is used, a capillary transfer may be best. Either capillary or electrical transfers can be performed with polyacrylamide gels.

When possible, use a gel system that was used successfully by other researchers to detect your protein of interest. Commercially available DNA retardation gels often work well for EMSA applications. Both the gel type and running buffer composition can influence how well an EMSA works. The following journal article provides an overview of some considerations often overlooked by researchers performing gel-shift assays: Roder, K. and Schwiezer, M. (2001), Biotechnol. Appl. Biochem. 33:209-214.

The LightShift™ Chemiluminescent EMSA Kit is composed of two sets of components that require different storage temperatures. One component set consists of the chemiluminescent substrates and various buffers that are stored at 4°C. The other component set consists of the control DNAs and various optimization reagents that are stored at -20°C. The EBNA extract must be maintained at -20°C or it will lose activity (proteins will degrade). Short-term storage (overnight) of the other kit components at temperatures ranging from room temperature to -20°C will not adversely affect kit performance.

Typically, the target duplex in EMSA is 20-35 bp long. Our scientists have successfully used DNA sequences as long as 60 bp. The actual binding sequence for protein:DNA interactions is frequently 10-15 bp. Longer probes can be used if the binding sequence is unknown or if multiple regulatory regions are being studied.

Yes. The Chemiluminescent Nucleic Acid Detection Module Kit (Cat. No. 89880) and LightShift™ EMSA Optimization and Control Kit (Cat. No. 20148X) are available separately.

The LightShift™ Chemiluminescent EMSA assay protocol, from making the DNA probe to visualization of the results, can be completed in approximately 5 hours.

The LightShift™ Chemiluminescent EMSA Kit is as sensitive as ³²P when optimized. In addition, the experiment can be completed in about 5 hours without the need for an overnight exposure often required when using ³²P. As with every system, some optimization may be required. The LightShift™ Chemiluminescent EMSA Kit has superior detection sensitivity compared to DIG detection systems.

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.

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.

The RNA Protein Pull-Down Assay is an in vitro technique for detection of protein-RNA interactions using labeled RNA as bait for protein. RNA is labeled with desthiobiotinylated cytidine and bound to streptavidin magnetic beads. The magnetic beads are then incubated with cell lysate to capture proteins that interact with the attached RNA Proteins are then gently eluted using biotin and detected by western or MS analysis. Because of the gentle elution (through use of desthiobiotin), RNA protein complexes may be captured and detected. The kit contains a pull-down control (RNA and antibody) that capture the correct protein from most lysates.

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) or RNA 3’ End Desthiobiotinylation Kit (Cat. No. 20163). Desthiobiotinylated cytidine is utilized for the pulldown assay to enable a gentle elution to capture protein-RNA complexes. Reagents and conditions are included in the kit to relax the RNA for better labeling efficiency. Additionally, suggestions are included in the instruction manual for optimization of the ligation reaction. An RNA control is included in the kit to assess kit labeling efficiency.

Chromatin immunoprecipitation (ChIP) assays identify links between the genome and the proteome by monitoring transcription regulation through histone modification (epigenetics) or transcription factor:DNA binding interactions. The strength of ChIP assays is their ability to capture a snapshot of specific protein: DNA interactions occurring in a system and to quantitate the interactions using quantitative polymerase chain reaction (qPCR). Chromatin IP experiments require a variety of proteomics and molecular biology methods including crosslinking, cell lysis (protein-DNA extraction), nucleic acid shearing, antibody-based immunoprecipitation, DNA sample clean-up and PCR. Additional techniques such as gel electrophoresis are usually used during optimization experiments to validate specific steps. See here for more details.

There are a few major differences between our agarose and magnetic ChIP kits. One is, of course, the beads. Both beads are blocked with different reagents. The magnetic beads are amenable to ChIP-seq as well as automation for the washing and elution steps. The second major difference is the chromatin preparation. Both kits use MNase to shear the chromatin into the appropriate sized fragments. The agarose kit uses a high-salt buffer to leak the nuclear contents after shearing, while the magnetic kit uses a quick sonication step to mechanically disrupt the crosslinked nuclei. The high-salt extraction can be very effective, however, if trouble is seen, the mechanical disruption can greatly improve results. The third major difference is the wash buffers. Each kit was optimized for its specific bead matrix (agarose or magnetic beads) to minimize background.

The Agarose ChIP Kit is not appropriate for use with ChIP-seq applications; however, the Magnetic ChIP Kit (Cat. No. 26157) can be used for ChIP-seq applications as the magnetic beads are blocked with a non-DNA containing buffer.

The advantages of enzymatic digestion include reproducibility of digestion, control of the reaction, and easy titration of the enzyme for each specific cell type. Our ChIP kits include a specially titrated and tested micrococcal nuclease that digests the DNA, minimizing variable results caused by the traditional method of sonication.

• The cycle threshold is the PCR cycle number where the fluorescent signal exceeds the background threshold level. This number is determined by your qPCR instrument and software.
• A typical signal from the rabbit IgG IP is usually ≥30 cycles while the typical signal from a positive control antibody IP (such as the RNA-polymerase antibody provided in the kit) is ~25 cycles.
• To be considered significant, a Ct difference of at least 3 is needed between the negative control (rabbit IgG) and your specific antibody.