LightShift EMSA Kit From Pierce

Our lab uses this kit to study the DNA-binding properties of different transcription factors involved in the regulation of aging using worms as a model system. We decided to employ the Pierce LightShift Chemiluminescent EMSA (Electrophoretic Mobility Shift Assay) Kit as an alternative to performing EMSAs with the radioactive isotope ³²P. This kit uses a non-isotopic method, utilizing biotin end-labeled DNA. The samples are subjected to gel electrophoresis on native polyacrylamide gels and transferred to positively charged nylon membranes. The biotin end-labeled DNA is detected using a Streptavidin-Horseradish Peroxidase Conjugate and the Pierce Chemiluminescent Nucleic Acid Detection Module.

The kit provides enough product for about 100 binding reactions and consists of: 10X Binding Buffer, Biotin-EBNA Control DNA, Unlabeled EBNA DNA, Epstein-Barr Nuclear Antigen (EBNA) Extract, Poly (dI-dC), 50% Glycerol, 1% NP-40, 1 M KCl, 100 mM MgCl₂, 200 mM EDTA and 5X loading buffer. Items that are not provided, but required, are positively charged nylon membrane, 5X TBE and a UV lamp.

The manual that comes with the kit is easy to follow and is suited for running mini-polyacrylamide gels. Conveniently, this kit provides the necessary positive control biotin DNA, negative control unlabeled DNA (competition) and extracts, eliminating the need to design the probe and make the extracts ourselves. With the control Epstein-Barr Nuclear Antigen (EBNA) System, we get very nice binding results that are easily checked using the control DNA.

We are using this EMSA kit to examine protein-DNA-binding activity in extracts prepared from wild type and mutants using worm nuclear extracts and biotin-labeled DNA probe which contains the DNA binding site of interest. The binding reaction using the kit’s binding buffer requires only 15 minutes. Previously, when I was performing EMSAs in a previous laboratory with ³²P labeled probes, the binding reactions took at least an hour. We incubate 1 ug of extracts with biotin-labeled oligonucleotide in binding buffer. The reaction samples are loaded and run on native polyacrylamide gels. The binding reactions are transferred onto PVDF membrane using the Bio-Rad semi-dry transfer apparatus, and then the DNA is cross-linked to membrane with a UV illuminator. The biotin-labeled DNA complexes are visualized using the Pierce Chemilumnescent Nucleic Acid Detection Module. Using this detection kit, we can get results in little over 30 minutes.

Although we were a bit skeptical regarding the effectiveness of the kit as compared to radioactive labeling, we have found that the sensitivity of this kit is comparable to a ³²P labeled probe. Although we had some initial problems mastering the technique with the kit and sample preparation, we have since been able to alleviate these problems. We have found that it is very important to use fresh extracts (frozen and thawed only once). After more than one freezing/thawing, we do not detect DNA-binding activity. Also, to prepare nuclear extracts, we use the homogenization technique cited in the following references: Stroeder et al, Dev. Biol. 163:367 and MG Hawkins et al, JBC 270:14666.