Pierce Biotechnology is a leader in offering products for the study of proteins and related research. Pierce offers kits and reagents for the detection and isolation of proteins, protein purification, protein labeling, interaction of proteins, and related studies. They also offer ELISA and array-based detection of proteins as well as Cellomics HitKit reagents for high content screening. They have provided consistent and reliable products to my laboratory throughout the years. This company also offers a very useful technical reference section on their website that answers a number of questions relating to protein science. The LightShift Chemiluminescent EMSA kit has been utilized in my laboratory for approximately 6 months, and while we are still working out the binding assay parameters, the kit has provided extremely clean blots during the optimization process.
The LightShift EMSA kit uses a nonisotopic method to study DNA-protein binding interactions. The technique is based on the fact that bound DNA and protein complexes run slower in a native polyacrylamide gel than unbound DNA thus resulting in a “shift” in migration of the bound DNA. The principle for LightShift EMSA Detection is similar to that of a Western blot and it utilizes a mini polyacrylamide gel (8 x 8 x 0.1 cm) which is one of the reasons this kit is very easy to work with. Biotin end-labeled duplex DNA is incubated with a protein extract and electrophoresed on a native gel. The DNA is then transferred to a positive nylon membrane, UV-crosslinked, probed with streptavidin-HRP conjugate and incubated with the substrate. The labeled DNA is then detected using X-ray film or a CCD camera. The whole protocol can be completed in one day, depending on the amount of time required for incubating the binding reaction. The good thing about the protocol is that once you have crosslinked the DNA onto the membrane, the labeled DNA can be detected at a later date.
The EMSA kit provides everything that is required to perform the assay except for the following obvious supplies/reagents: Biotin end-labeled (3’ or 5’) DNA as well as unlabeled DNA, desired protein or nuclear extract, UV lamp or crosslinking device, materials and equipment used for Western blots including positively charged nylon membranes, and a waterbath to incubate the binding reactions. The DNA of interest can be synthesized with an end-labeled biotin molecule or the labeled DNA can be prepared yourself using the Biotin 3’-end DNA labeling kit from Pierce (catalog number 89818). Internal biotin labeling is not recommended since it can interfere with DNA-protein binding. Also, it is suggested by the company to individually label the single stranded DNA first before annealing the two strands together. I have used the Pierce kit to label my DNA with biotin and it has worked well with the EMSA kit. I have also known people to have their DNA labeled with biotin by the manufacturer of their oligos and they have had success with this as well. The kit contains a 10X binding buffer, a control system consisting of a biotin-EBNA DNA and unlabeled DNA as well as the EBNA protein extract, loading buffer, and many other components that are used to optimize the binding reaction including Poly(dI.dC), a nonspecific DNA competitor. The kit also contains the Chemiluminescent Nucleic Acid Detection Module which contains the buffers, substrate, and streptavidin-HRP conjugate needed to detect the biotin-labeled DNA on the nylon membrane.
The protocol is fast and extremely easy to follow. Briefly, while the native polyacrylamide gel in 0.5x TBE is pre-running at 100 volts for 30-60 minutes, the binding reactions are incubated for 20 minutes or more at room temperature or whatever temperature is appropriate for your reaction. Three different binding reactions are suggested for each sample: Labeled DNA (probe) alone, probe with protein extract, and probe with protein extract along with excess unlabeled DNA. This last reaction is to show competition and specific DNA-protein binding since the unlabeled DNA should bind more protein and thus no or little shift should be detected. Once the binding reactions are complete, loading buffer is added to each and the reactions are loaded onto the pre-run gel. The gel then runs until the bromophenol blue dye migrates 2/3 to 3/4 of the length of the gel. Once complete, the proteins from the gel are transferred to the nylon membrane for approximately 30 minutes at 100 volts in a standard transfer apparatus. Once complete, the DNA is crosslinked to the membrane for 15 minutes using a transilluminator with a 312 nm bulb. Other equipment that can be used for crosslinking includes a UV- light cross-linker or a hand-held UV lamp. The biotin-labeled DNA is then detected by chemiluminescence following the protocol and using the buffers provided in the kit. The detection of the labeled DNA takes approximately 1 hour. I then expose the membrane to X-ray film. The film is then developed according to manufacturer’s instructions. A CCD camera can also be used.
The LightShift Chemiluminescent EMSA Kit by Pierce Biotechnology has been a useful tool in my laboratory for the study of aptamer-protein binding interactions. Even though I am still working through the optimization of the binding reaction, the kit provides such clean results that it makes working out the kinks much more enjoyable. I recommend this kit for anyone interested in visually detecting protein and DNA binding while determining the specificity of the binding interaction. Since the kit is non-radioactive and uses the mini polyacrylamide gels, the assay is extremely fast and user friendly. I recommend using smaller volumes of detection reagent than the protocol suggests because I believe the protocol calls for an excessive volume – just make sure to cover the membrane. Using a small container that fits the membrane for the detection will save reagent and thus, money. I believe this kit will provide the answers any researcher is asking in regards to DNA-protein interactions. I recommend it to everyone, but especially those who currently use the radioactive EMSA technique since it is so much faster and produces cleaner results.