A Detailed and Radioisotope-free Protocol for Electrophoretic Mobility Shift Assay (EMSA)

[Abstract] To comprehensively characterize the functions of a transcription factor (TF), it is required to analyze the interaction of this TF with its targeted loci. Several methods such as β -glucuronidase (GUS) or luciferase reporter, yeast one-hybrid (Y1H), chromatin-immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA) assays have been developed. Of these, EMSA is an in vitro method which can prove the direct interaction between TF and targeted DNA fragment. In the present protocol, DNA probes are labeled with Biotin. Therefore, it is safer for researchers when they do not need to use radioisotope-labeled probes. In addition, this protocol is to provide a detailed procedure for a successful EMSA assay. The interested recombinant protein can be mixed with putative-targeted DNA probes for a binding reaction at room temperature (25 °C). Afterward, the reaction mixture can be run in a native polyacrylamide gel and transferred to a positively charged nylon membrane. Finally, the results can be detected and visualized via the biotin-streptavidin chemiluminescence.


Note: Basically, the oligo can be delivered in dried form (in a micro-tube). Based on the amount of dried oligo and the company's instruction, add molecular biology-grade water (or Milli-Q purified water) to each oligo tube to obtain the final concentration (100 µM).
2. Place the micro-tube in a heat-block at 95 °C for 5 min.
3. Afterward, turn off the heat-block. Remove the metal rack containing the micro-tube and let all cool down at room-temperature.
Note: At this step, an alcohol thermometer can be placed in the metal rack to track the decrease of the temperature. The double-strand DNA can be used when the temperature is around 25 °C.

Store at -20 °C for further experiments.
Note: The labeled DNA probes should be aliquoted before storing to avoid freeze-thaw cycles.
B. Native polyacrylamide gel (5%) preparation 1. Prepare 5% native polyacrylamide gel as follows (    4. Next, add 1 µl of the unlabeled DNA probes (or molecular biology-grade water) (as shown in Table 5), gently mix up and let the reaction at room-temperature (25 °C) for 15 min. 2. After electrophoresis, carefully transfer the gel onto a filter paper, foam pad, and the black-side of the gel holder cassette as demonstrated in Figure 1. Place the membrane (from Step E1) on the gel. Carefully remove the air-bubbles between the membrane and the gel (can use the Bio-Rad roller or can roll a measuring pipette on the membrane). Next, place one more filter paper and a foam pad on the membrane. Lock the gel holder cassette and transfer this to the buffer tank containing the electrode (Figure 1). Fill up the buffer tank with 0.5x TBE buffer. Next, cover the lid of the buffer tank and start the transferring.
3. Transferring conditions: stable current and 100 V at room-temperature in 0.5x TBE buffer. And the transferring time is 30 min.  3. Subsequently, the membrane can be used for detection or stored at room-temperature for a few days.

G. Detection
The Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific) is used and the detection step follows the company's instruction.
Note: For the final detection step, a bio-imaging system equipped with a charge-coupled device (CCD)-camera or even conventional method using X-ray film can be used.

Data analysis
The obtained result can be exhibited as Figures 2 and 3. As shown in Figure 3, the band indicating the shifted probe was observed when the interested protein (an MYB transcription factor) binds to its targeted DNA. In the neighbor reaction, the unlabeled probe (300x) was added and these unlabeled DNA strongly competed with the labeled one (1x) resulting in the abolishment of the shifted band. Besides, there were no shifted probes in the negative control assay (the interested protein and non-targeted DNA).

1 M and 100 mM DTT
To prepare 10 ml of 1 M DTT a. Dissolve 1.545 g of DTT in around 5 ml of molecular biology-grade water b. Next, add more molecular biology-grade water to achieve 10 ml c. Mix 1 ml of 1 M DTT with 9 ml of molecular biology-grade water to achieve 10 ml of 100 mM DTT d. Aliquot 1 ml to each micro-tube and store at -20 °C

10x TBE buffer
To prepare 1 L of 10x TBE buffer a. Mix Tris base (108 g), boric acid (55 g), and EDTA (disodium salt) (9.3 g) with molecular biology-grade water (~500 ml) b. Next, add more molecular biology-grade water to achieve 1 L c. Autoclave for 20 min and this 10x TBE buffer can be stored at room-temperature