Q. What Is an Electrophoretic Mobility Shift Assay (EMSA)?
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FAQs LightShift® Chemiluminescent EMSA Kit
Q. What is an electrophoretic mobility shift assay (EMSA)? A. EMSA (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 discreet bands within the gel using chemiluminescence or radioisotopic detection.
Q. What are the applications for the LightShift® Chemiluminescent EMSA Kit? A. The LightShift® Chemiluminescent EMSA Kit uses proprietary chemiluminescent detection technology to perform nonradioactive EMSA or supershift assays.
Q. How does the assay work? A. 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.
Q. How does the sensitivity of the LightShift® Kit compare to systems using DIG or 32P? A. The LightShift® 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® Kit has superior detection sensitivity compared to DIG detection systems.
Q. How long does it take to complete this assay? A. The LightShift® protocol, from making the DNA probe to visualization of the results, can be completed in approximately 5 hours.
Q. Can kit components be purchased separately? A. Yes, the Chemiluminescent Nucleic Acid Detection Module could be purchased separately (Product# 89880).
Q. Does Pierce sell biotinylated probes? A. No. Pierce does not sell biotinylated probes. However, for a small fee, many companies that synthesize custom oligonucleotides will add a 5´ biotin during synthesis. You can prepare biotinylated probes using Pierce’s Biotin 3´ DNA Labeling Kit (Product # 89818).
Q. How should I prepare my biotinylated DNA? A. 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 (Product # 89818). Internal biotin labels may inhibit binding of the DNA binding protein and are not recommended.
Q. Is there a limit to the length of target DNA I can use with this kit? A. Typically, the target duplex in EMSA is 20-35 bp long. Pierce scientists have successfully used DNA sequences as long as 60 bp. The actual binding sequence for a protein:DNA interaction is frequently 10-15 bp. Longer probes can be used if the binding sequence is unknown or if multiple regulatory regions are being studied.
Q. Can I use agarose gels or must I use acrylamide gels? A. EMSAs may be performed with either polyacrylamide or agarose gels depending upon the resolution requirements of the customer's 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.
Q. Are there any considerations I should be aware of when selecting the type of gels to use? A. 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.
Q. How should I transfer my completed gel-shift to a membrane? A. Transfer to nylon membrane can be performed using an electrophoretic transfer apparatus or using traditional capillary transfer methods. Both methods are provided in the kit instructions.
Q. What type of membrane should I use? A. Positively charged nylon membrane must be used. Pierce scientists use Biodyne B Membrane (Product # 77016).
Q. How much of a “shift” should I expect to see? A. 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.
Q. What if I don’t see a shift? A. The reaction may need to be optimized. System optimization can be achieved by adding various components supplied with the kit 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.
Q. My experiment worked when I used 32P, what is wrong? A. The LightShift® Kit is used to detect a biotinylated probe in an EMSA. The binding conditions for each experiment must be optimized for the proteins tested. If an experiment was working when 32P labeled probes were used, then use the same binding conditions from those previous experiments and only substitute the biotinylated probe in place of the 32P labeled probe. While commonly used reaction components are provided with the LightShift® Chemiluminescent EMSA Kit, these reagents may not be applicable in all situations.
Q. What if I see multiple shifts when I analyze the EMSA? A. 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).
Q. How do I make the nonspecific bands go away? A. Nonspecific bands may be reduced or eliminated by optimizing the binding reactions. Strong nonspecific bands can often be mistaken for a positive result but will not be blocked by the addition of cold competitor probe. The following are examples of changes that MAY help reduce nonspecific bands: •Reduce the amount of protein extract in the binding reaction •Increase the amount nonspecific competitor DNA [i.e., poly(dI-dC)·poly(dI-dC)] • Use a different nonspecific competitor DNA [i.e., sonicated salmon sperm DNA or poly(dA- dT)·poly(dA-dT)] •Preincubate the protein extract with nonspecific competitor DNA before adding the biotinylated probe. •Shorten or redesign the probe used in the experiment.
Q. Why is my shift not blocked by cold probe? A. The shift may be caused by nonspecific binding or a DNA binding protein different than the one being tested.
Q. What are the optimal reaction conditions for my protein? A. The binding conditions for each experiment must be optimized for the proteins tested. Journal articles are an excellent source for information on how to set up EMSA binding reactions. If an article describes the use of 32P labeled probes, repeat the binding conditions from that experiment, but substitute a biotinylated probe in place of the 32P labeled probe. Although reaction components are provided with the LightShift® Kit, these reagents may not be suitable for individual applications and may be adjusted as needed.
Q. What is the purpose of adding unlabeled DNA to the binding reaction as a control? A. 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.
Q. Can the LightShift® Kit be used to perform supershifts? A. Yes, the LightShift® 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. For examples of how the LightShift® Kit was used to detect supershifts, see the following: 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.
Q. What is a “supershift”? A. A supershift assay is a method for positively identifying a protein:DNA interaction on an EMSA. An 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” cause 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.
Q. How much protein do I need for each reaction? A. 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 nonspecific bands.
Q. Can I probe for the proteins by performing a Western blot? A. This has not been tested, but may be possible. A better alternative would be to perform a DNA binding protein pull-down assay using a probe. The following journal article is a good example of how the LightShift® 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.
Q. If the kit has been improperly stored (i.e., at room temperature, -20°C or +4°C), will it still work correctly? A. The LightShift® 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.
Q: How do I prevent the gel from sticking to the membrane after the transfer step? A: The gel sticking to the membrane typically results from one of two problems. First, make sure to soak the membrane in 0.5X TBE for at least 10 minutes prior to transferring the binding reactions. Second, overheating during the transfer may cause the gel to begin to melt. To prevent overheating cool the 0.5X TBE transfer buffer to approximately 10C with a circulating water bath. Alternatively, perform the transfer in a cold room ( 4C). If the gel has already stuck to the membrane soak the gel/membrane in 0.5X TBE using gentle agitation to separate the gel. Do not scrape the gel off the membrane as this may damage the gel or result in high background.
Q: Is the LightShift Chemiluminescent Gel Shift Kit as sensitive as radioactive gel shifts? A: A direct comparison of a radioactive (probes end labeled with 32P) gel shifts to LightShift chemiluminescent detection using the Oct-1 consensus sequence demonstrated equivalent sub- femtomole (~50 attamoles) detection and an identical shift pattern.
Q: What transcription factor systems has Pierce evaluated with LightShift system? A: The LightShift system has been demonstrated to detect the AP1, NF-B and Epstein Barr Nuclear Antigen 1 (EBNA) proteins binding to their respective DNA consensus sequences.
Q: How long does it take to complete this assay? A: The entire assay from DNA labeling to detection of EMSA products on the membrane takes less than five hours.
Q: Is the LightShift Chemiluminescent Gel Shift Kit compatible with antibody supershift assays? A: Supershift assays have been evaluated and detected using the Oct-1 DNA consensus sequence
Q: What if I see multiple shifts when I analyze the EMSA? A: It is possible to see multiple shifts in an EMSA. This is most likely due to the DNA binding to multiple forms of the protein such as a monomer vs. a dimer, or perhaps due to multiple factors binding to the same site or to multiple binding sites within the target DNA (especially common with longer duplexes).
Q: Can I use agarose gels or must I use acrylamide gels? A: EMSAs may be performed with either polyacrylamide or agarose gels depending upon the resolution requirements of the customer's system. Traditionally, 4-6% polyacrylamide gels are used. If agarose is used, a capillary transfer may be best. Either capillary or electrical transfers can be performed with acrylamide gels.