Shop online for a wide selection of Thermo Scientific LightShift Chemiluminescent EMSA Kit Identify and characterize protein-DNA binding interactions by EMSA. 14 Mar A review of the LightShift Chemiluminescent EMSA Kit From Pierce. Unbiased reviews by scientists available at 21 Feb A review of the Pierce’s Lightshift Chemiluminescent EMSA Kit. Unbiased reviews by scientists available at
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The protocol is fast and extremely easy to follow. This membrane is developed using the Pierce Kit, and from start to finish can take as little as five hours. Until conception of the EMSA protein: The sample is run on a gel where the unbound labeled DNA is separated from the slower migrating transcription factor bound DNA.
Pierces Lightshift Chemiluminescent EMSA Kit
J Exp Biol— Cornelussen, R. Once complete, the proteins from the gel are transferred to the nylon membrane for approximately 30 minutes at volts in a standard transfer apparatus. The time and money saved using this kit will more than make up lit the initial need to optimize experiments. Mol Micro 56 2— Oltra, E.
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. Biotin end-labeled duplex DNA is incubated with a nuclear extract or purified factor and electrophoresed on a native gel. Highlights Excellent for detecting low-abundance proteins in nuclear extracts Sensitivity that surpasses radioactive and digoxigenin methods Includes EBNA control system to help new users develop a working assay and understand the methods used to confirm binding interaction specificity Compatible with previously-established binding conditions for popular DNA: I did notice how many more gel shifts I could do.
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Pierce offers kits and reagents for the detection and isolation of proteins, protein purification, protein labeling, interaction of proteins, and related studies. DNA binding reactions, a control set of DNA and protein extract to test the kit system, stabilized streptavidin-HRP conjugate to probe for the biotinlabeled DNA target, and lightsuift exceptionally sensitive chemiluminescent substrate module for detection.
The EMSA technique is based on the observation that protein: The whole protocol can be completed in one day, depending on the amount of time required for incubating the binding reaction. Since the kit is non-radioactive and uses the mini polyacrylamide gels, the assay is extremely fast and user friendly. Because the rate of DNA migration is shifted or retarded upon protein binding, the assay is also referred to as a gel shift or gel retardation assay. Endocrinology 7 Equivalent exposures using the two chemiluminescent kits showed that the sensitivity of the LightShift Cgemiluminescent Kit was approximately eight-fold greater than that of the digoxigenin kit.
Tuesday, February 21, Internal biotin labeling is not recommended since it can interfere with DNA-protein binding.
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J Biol Chem 35— Biotinlabeled target duplexes ranged in size from bp. I took advantage of the longer shelf life of the labeled probes, and streamlined the EMSAs by producing all the probes I would need for months. Binding reactions were equivalent in that 20 fmol duplex was incubated with 6. This kit is also more sensitive than radioactive and digoxigenin methods. The interaction of proteins with DNA is central to the control of many cellular processes including DNA replication, recombination and repair, transcription, and viral assembly.
Chemiluminescent Nucleic Acid Detection Moduleavailable separately. The Good This kit replaces expensive and hazardous radioactive gel shifts without losing sensitivity.
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Other equipment that can be used for crosslinking includes a UV- light cross-linker or a hand-held UV lamp. Clin Cancer Res 10 This kit is also compatible with super shifts although I personally have no experience with this.
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. Stay informed by signing up for our eNewsletters and start receiving. One technique that is central to studying gene regulation and determining protein: I recommend this kit for anyone interested in visually detecting protein and DNA binding while determining the specificity of the binding interaction. In addition to the hazardous materials associated with these probes, the cost of the radioactive material to make new probes every couple weeks is an important consideration when choosing this method.
To make these radioactive DNA probes is an expensive and long procedure that is complicated by the probes short shelf life of about a week.
Once their probes are annealed and purified they last for months not weeks and they cost a fraction of radioactive probes. The detection of the labeled DNA takes approximately 1 hour. Exposure time was 30 seconds with X-ray film. With this knowledge, the complex signal cascades in the cell can be understood in regards to which proteins will be actively transcribed due to an experiment response.
In addition, the transfer from gel to membrane and following development require additional care to ensure high quality data. All you need to perform the assay are purified DNA target that has been end-labeled with biotin, the protein extract you wish to test, nylon membrane and basic electrophoresis equipment.