We use the Pierce Chemiluminescent Nucleic Acid Detection Module to detect biotin-labeled nucleic acids containing the putative binding site of our protein of interest, Heat Shock Factor (HSF), in Electrophoretic Mobility Shift Assays (EMSAs), also known as gel-shift assays. I am using this detection kit in conjunction with the Pierce LightShift EMSA Kit. The LightShift EMSA Kit provides the reagents necessary to generate a biotin-labeled probe and run the EMSA assay. We then use the Chemiluminescent Nucleic Acid Detection Module for detection. We use these kits to examine different transcription factor, DNA-binding activity in extracts prepared from
C. elegans to further understand transcriptional regulation in aging. The Nucleic Acid Detection Module uses an enhanced luminal horseradish peroxidase (HRP) substrate with optimized blocking and washing. This kit produces results similar to those obtained with a
32P probe, but without radioactive exposure and handling. The kit includes stabilized Streptavidin-HRP Conjugate, Blocking Buffer, 4X Wash Buffer, Substrate Equilibration Buffer, and chemiluminescent substrate (made by mixing 1:1 luminol/enhancer solution and stable peroxidase solution). One kit contains enough reagents for about 1000 cm2 of membrane.
Before using the reagents, they should be warmed to room temperature. The membrane is blocked for 15 min in Blocking Buffer, followed by incubation for 15 min with the stabilized Streptavidin-HRP Conjugate/Blocking Buffer. The membrane is then washed and incubated for 5 min in Substrate Equilibration Buffer. During this step, the chemiluminescent substrate working solution is prepared by mixing luminal/enhancer with peroxidase solution. The membrane is incubated with this working solution for 5 min, followed by exposure to X-ray film. For EMSAs, we were getting weak signals for our protein-DNA complexes (related to our protein extract) so we had to expose the film much longer than recommended.
Many of our initial problems with weak bands or no bands were due to our extract preparation. We have partially alleviated that problem using published protocols involving homogenization (Stroeder et al, Dev. Biol. 163:367 and MG Hawkins et al, JBC 270:14666) and by extending the exposure time on film. With these modifications, we are now able to consistently detect our transcription factor-DNA complexes, at least for extracts prepared from wildtype worms.
We have also been able to use these luminol/enhancer reagents for detection of Western blots, although signal enhancement resulted in higher background problems. We were not getting any signal or very weak signal for transcription factors involved in the stress response when using the regular, cheaper ECL reagent (Pierce ECL Western Blotting Substrate), but we were able to get very good signal using this kit. To alleviate the problem with high background, washing the membrane briefly with water right after incubating with the working solution has been helpful.
Overall, the Pierce Chemiluminescent Nucleic Acid Detection Module Kit is a good product to use in conjunction with the Pierce LightShift EMSA Kit. It can also be used to enhance signals on difficult Western blots. It is very easy to use, requires very little time and the manual is very easy to follow. On the negative side, it is expensive and the small amount of reagents that come with the kit allows for limited analyses.