Pierce’s Lightshift Chemiluminescent EMSA Kit

Pierce’s Lightshift Chemiluminescent EMSA Kit
Transcription factors control the production of proteins and understanding which transcription factor affects which protein’s production is essential. 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.

Looking at active transcription factors in a cell can tell the experimenter what proteins are being produced and more generally what signals are being activated or deactivated in a cell. Traditionally, electromobility gel shift assays (EMSA) using radioactive DNA probes were the most common method for determining the activity of a transcription factor. Briefly, radioactively labeled double stranded DNA with the binding sequence of a specific transcription factor is incubated on ice with a nuclear protein extract (or recombinant transcription factor). The sample is run on a gel where the unbound labeled DNA is separated from the slower migrating transcription factor bound DNA.

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. 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.

Luckily, there is another option. The Pierce Lightshift chemiluminescent EMSA Kit contains all of the essential supplies to get up and running except for the DNA probes. Most companies that synthesis custom oligonucleotides will add a 5‘ biotin for a small fee. Once their probes are annealed and purified they last for months not weeks and they cost a fraction of radioactive probes. The method behind this biotin strept-avidin HRP EMSA is essential the same as a chemiluminescent Western Blot. The EMSA is run under the same conditions explained previously, and then the gel is transferred to a membrane. This membrane is developed using the Pierce Kit, and from start to finish can take as little as five hours.

When I joined my current lab, they had just started the transition from radioactive to biotin labeled EMSAs. I ran a few radioactive gels, and when I switched I noticed a real difference. The Pierce kit is just quicker and less expensive then the radioactive option, and I did not notice a difference in sensitivity. I did notice how many more gel shifts I could do. 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. And then after optimizing the shifting conditions for each transcription factor, I could get results that much quicker. Besides the time taken to optimize the biotin experiments, a real drawback to this method is that the biotin can affect the binding of the transcription factor to the DNA probe. To minimize this the biotin label is added to the 5 ` or 3 ` end of the DNA probe. This kit is also compatible with super shifts although I personally have no experience with this.

Jeffrey Perry
Lab Manager
University of Michigan

  • <<
  • >>
Pierce’s Lightshift Chemiluminescent EMSA Kit
The Good

This kit replaces expensive and hazardous radioactive gel shifts without losing sensitivity.

The Bad

Electromobility gel shift assays (EMSA) are unique to the protein of interest, and therefore optimization of shifting conditions will be required. The nature of the biotin labeled probe create the possibility of the biotin blocking or restricting the transcription factor from binding. In addition, the transfer from gel to membrane and following development require additional care to ensure high quality data.

The Bottom Line

The time and money saved using this kit will more than make up for the initial need to optimize experiments.

Comments