The overall of this protocol is to analyze RNA protein interactions using horizontal native gel electrophoresis. This method can help answer key questions in RNA biochemistry, such as defining the characteristics of specific RNA protein interactions. The main advantage of this technique is that it allows complex formation to be monitored multiple times during electrophoresis.
For the horizontal gel apparatus, use a gel box with a 27 centimeter by 21 centimeter casting tray and a capacity for two 24 well combs. This set up provides a total of 48 samples that can be analyzed simultaneously. Prepare two liters of 1x TBE running buffer by diluting 400 milliliters of 5x TBE with 1, 600 milliliters of water and mixing.
Place the TBE buffer in the cold room to cool. Prepare the following reagents following standard procedures, 40%acrylamide best 19:1, 5x TBE, temed and 10%ammonium per sulfate. To make a 10%native acrylamide gel, add the following to a 500 milliliter flask:80 milliliters of 5x TBE, 100 milliliters of 40%acrylamide and water to a final volume of 400 milliliters.
Add four milliliters of 10%ammonium per sulfate and 400 microliters of temed and mix well. Pour the mix into the horizontal casting tray and allow the gel to polymerize in the fume hood for 30 minutes. The polymerized gel will be approximately two centimeters thick and a thin layer of liquid will remain on top of the gel.
Place the gel into the gel box and take it into the cold room. Next, pour the liquid off and rinse with running buffer. Remove the combs carefully, add two liters of cold running buffer to the gel box and then use a syringe to rinse the wells with running buffer.
Pre-run the gel at 120 volts for one hour at four degrees celsius in a cold room. While the gel is being prerun, prepare the binding reactions. Add the following binding reaction components to each RNA free microcentrifuge tube in this order:five microliters of 10 millimolar BSA, five microliters of 20 millimolar DTT, five microliters of 10 millimolar yeast tRNAs, 10 microliters of 5x binding buffer, bicaudal-c protein to a final concentration of 250 nanomolar in 50 microliters, DEPC-treated water to a final volume of 45 microliters and five microliters of fluorescently labeled RNA substrate.
Close the tubes and flick gently to mix, centrifuge the tubes briefly and incubate in the dark for 30 minutes at room temperature. When the binding reactions are complete, add 15 microliters of 50%glycerol to each reaction and mix gently. Carefully load 30 microliters of each reaction into the wells of the gel, the amount of sample that can be loaded into a single well varies depending on the thickness of the gel and the size of the wells.
The power supply to the gel apparatus, switch on the power, adjust the voltage to 120 volts and run the gel in four degree celsius. To prevent damaging or bleaching of the fluorescently labeled RNA, conduct electrophoresis in the dark. Electrophoresis times will vary depending upon the specifics of the RNA protein complex being analyzed.
A major advantage of the horizontal electrophorated mobility shift assay analysis, is that electrophoresis can be stopped in the gel image prior to completion of the run. Perform analysis with any instrument designed for detecting an imaging fluorescent substrates. Adjust the imager settings, for fluorescently labeled ligands, use the following settings.
Fluorescein wavelength of 473 nanometers, photomultiplier voltage of 750 volts and pixel size of 100 micrometers. Scan the gel to capture the image. After imaging, the gel can be placed back into the apparatus and electrophoresis can be continued for longer times.
The binding of the xenopus bicaudal-C or Bicc1 protein to a fluorescently labeled Cripto1 mRNA containing a Bicc1 binding site, was analyzed by both vertical and horizontal native gel electrophoresis, as a negative control, Bicc1 was mixed with a fluorescently labeled RNA derived from CyclinB1 mRNA. It was readily apparent using either method, that Bicc1 binds to the Cripto1 RNA and forms a specific complex. However, the Bicc1, Cripto1 complexes were significantly more distinct when analyzed with the horizontal gel, facilitating their detection and allowing the discrimination of protein RNA complexes from unbound RNA.
After imaging, the gel was returned to the gel apparatus, electrophoresis for an additional three and a half hours, and reimaged. As shown here, the complexes had migrated further and were still readily detectable, indicating their stability. Once mastered, this technique can be done in four to five hours, if it is performed properly.
After watching this video, you should have a good understanding of how to analyze RNA protein interactions using horizontal native gel electrophoresis.
