Electrophoretic Mobility Shift Assay (EMSA) is a classic method for calculating dissociation constants (Kd) between a nucleic acid binding protein and its target sequence. Varying amounts of the protein of interest is incubated with a nucleic acid labeled with a radioactive isotope for visualization. The reactions are allowed to reach binding equilibrium, and then ran through a non-denaturing polyacrylamide gel to separate bound from unbound nucleic acid. In an ideal case, this will result in two bands in the gel: a higher band consisting of the protein bound to the nucleic acid, and a lower band band consisting of only nucleic acid. The Kd can then be calculated by plotting the fraction bound (the signal from the top band over the signal from the total top and bottom bands) against the protein concentration. The Kd is the point in the line where half of the nuclei acid is bound. For a nice review and discussion of the many variations of this technique, see “Nat. Protoc. 2007; 2(8): 1849-61“.
As an alternative technique, we will use Microscale Thermophoresis (MST) to determine our binding affinity. This is a relatively new technique that employs the phenomenon of thermophoresis, where molecules move through a temperature gradient. This phenomenon is dependent on size, charge, and solvation entropy of the molecule. Therefore, a binding event, such as a transcription factor binding its target nucleic acid, will drastically change these characteristics and therefore its movement along a temperature gradient. After fluorescently labeling the nucleic acid, an instrument developed by NanoTemper Technologies is able to observe this movement through the temperature gradient. By altering the protein concentration, similar to EMSA, and observing the thermophoresis of the nucleic acid, a Kd can be inferred. This is a relatively simple and quick method for getting the same information as an EMSA. At some point, there will be a detailed discussion of this technique.
We will be using both of these techniques to determine the binding affinity of the Gal4-p53 transcription factor with the target DNA sequence: TCCGGAGGACTGTCCTCCGGC. The EMSA experiment is largely based on “Biochem. and Molec. Bio. Education Vol. 40, No. 6, pp. 383-387“. The Gal4-p53 protein, as well as a Cy5 labeled DNA jairpin with the Gal4 binding site, will be provided for you.
We will 1) perform the EMSA, and 2) perform the MST experiments. The MST experiment will be done on a per-group basis with the scientists from NanoTemper.
Outline:
A. Classic EMSA (updated protocol)
- Prepare the gel
- Prepare EMSA reactions
B. Appendix: Buffers
A. Classic EMSA
For the EMSA, the DNA has already been prepared for you. In order to visualize the DNA on the gel, it need to be either radioactively or fluorescently labelled. Here we will use the fluorescent label, Cy5.
When loading the gels, the power needs to be on to quickly draw the DNA and DNA-protein complexes into the gel. Otherwise, the complexes at equilibrium may be diluted and dissociate prior to entering. Once the gel, the DNA-protein complexes are thought to stay associated due to the “caging affect”, whereby the gel matrix prohibits diffusion of any dissociated protein. Since the DNA and protein are stuck in such a close proximity, they are thought to immediately re-bind rather than separate which would cause a smear throughout the whole lane.
- Prepare a gel for the EMSA experiment
- Prepare the EMSA reaction
i. Mix the following in a 15 mL conical to make the gel mix: 1.2 mL 10X Tris-Glycine Running Buffer, 1.2 mL 50% glycerol, 2 mL 30% acrylamide, and bring to a final volume of 12 mL. Keep the solution on ice.
ii. Assemble the gel casting apparatus. Wash the plate with a 1.5 mm spacer and a front cover plate with detergent and water, and then rinse with 95% ethanol.
iii. Place the front cover plate over the spacer plate and fasten with the green casting frame. The bottoms of the plates need to line up very closely of the gel will leak. Place the secured plates in the gel casting stand.
iv. When everything is ready to pour the gel, add 67.7 uL 10% APS and 16.5 uL TEMED to your gel mix. This will begin the polymerization of the gel. Pipet the gel mix into your plate until the gel mix is near to the top, and then insert the 10 well 1.5 mm comb. Wait at least a half hour until the gel polymerizes.
i. Add 1 mM DDT to a 1 mL aliquot of Buffer B. Make the following Gal4-p53 dilutions in Buffer B: 400 nM, 40 nM, 4 nM. The stock solution is 25 uM or 1 mg/mL (protein is about 40 kDa). Since the protein binds as a dimer though, the working concentration is 12.5 uM.
ii. On ice, mix the following reactions (Add the DNA in Buffer A last).
iii. Incubate the reactions at RT for 30 min.
iv. Load 18 uL of each reaction on the gel while it is running (should have been running at 100 V for at least 30 min). Again, be careful not to touch the buffer or you mar be electrocuted
v. After about 45 min, carefully remove the gel from the apparatus. Slowly lift one plate so as to not rip the gel. It should stick to only one plate.
vi. Use the Typhoon Imager to image the gel.
B. Appendix: Buffer
Buffer A
20 mM HEPES
8 mM MgCl2
1 mM DTT (Add fresh)
50 uM ZnCl2
0.2 nM DNA
Buffer B
20% glycerol
20 mM Tris-HCl
100 mM KCl
1 mM DTT (Add fresh)
50 ug/mL
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