Analysis of RNA-peptide interactions by PACE
The polyacrylamide coelectrophoresis (PACE ) assay is a relatively recently developed method for quantitatively analyzing the interaction of proteins and peptides with DNA or RNA. This experiment is derived from the "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Analysis of RNA-peptide interaction experiments using PACE
Principle
The polyacrylamide coelectrophoresis (PACE ) assay is a relatively recently developed method for quantitatively analyzing the interaction of proteins, peptides and DNA or RNA.
Materials and Instruments
Low Radioactive Labeling RNA Substrate Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
TBE Gel Storage Buffer Electrophoresis Buffer Acrylamide Storage Solution Ammonium Persulfate Thomas Blue
Polyacrylamide Gel Electrophoresis Equipment PACE Gels Chromatography Paper Plastic Film Gel Dryers Dark Boxes
1. standard polyacrylamide gel electrophoresis equipment, including power supply, electrophoresis tanks, clamps, and hot bath (1/8-inch thick aluminum plate).
2. wide and short gels, because wide gels can contain more concentration gradients and short gels can reduce the amount of peptide needed per polyacrylamide strip, the typical size is 24 cm X 36 cm or 26 cm X 36 cm ( h X w ), the actual size of the gel is not critical, this procedure can be applied equally well to other sizes of gels in specific applications.
3. For PACE gels, the combs and spacers are cut, as depicted in Figure 11.5, and the cut gel can contain up to 10 peptide concentrations. 
4. 10 ml Luer syringe, 25 gage needle, gloves and paraffin membrane.
5. low-level radioactive marker.
6. chromatography paper, plastic membrane and gel dryer.
7. dark boxes, x-ray film and developer.
8. instruments for quantification of radioactive substances.
9. 5X TBE gel storage buffer and electrophoresis buffer: 450 mmol/L Tris base, 450 mmol/L boric acid, 10 mmol/L EDTA (pH 8.0).
10. 40% acrylamide storage solution (29:1, acrylamide: methacrylamide).
11. 10% ammonium persulfate (API aqueous solution (m/V), 99% TEMED.
12. 32P-labeled RNA substrate, PACE can use 3', 5', or internally labeled RNA. RNA samples can be prepared using standard procedures, but if only trace concentrations of RNA are to be sampled onto the PACE gel, high radioactivity (> 200 dμm/fmol) is required.
13. Prepare and purify peptides or proteins. Because the PACE assay is very sensitive, high purity and precise concentration of peptides is the most essential requirement. Peptides are generally purified by reverse high-performance liquid chromatography (PR-HPLC) or other high-precision purification methods, and protein or peptide solutions are serially diluted with water or a suitable storage buffer. The storage solution is usually formulated as 100X and diluted 100-fold at the time of use.
14. RNA samples are diluted into 6X of Type III Sampling Dye: 0.24% Bromophenol Blue, 0.24% Xylene Cyanine FF, 30% glycerol in water.
15. Kaomas Brilliant Blue Protein Staining: 0.24% Kaomas Brilliant Blue, 45% aqueous 45% methionine in 10% glacial acetic acid, used to calibrate the time of PACE electrophoresis.
Test Methods
1. Detection
(1) Wash each gel plate carefully with detergent and rinse with water, and then drench with ethanol. Ideal siliconized plates can ensure the gel mixture is evenly distributed after perfusion. Install the gel plate, comb, and spacer as shown in Figure 11.6, rotating it 90°. Slide out the upper spacer(s) to create a gap for perfusion, draw a line between the tops of the B and P relative to each other, mark the height of perfusion for each peptide concentration of gel, label each lane and record the concentration of peptide storage solution used. 
(2) Prepare 200 ml of gel mixture: 0.5X TBE, 15% ( 29:1) acrylamide, 0.02% ammonium persulfate (20 ml 5X TBE stock solution, 75 ml 40% acrylamide stock solution, 400 μl 10% AP, 104.6 ml redistilled water). Add salts ( Na+, K+, Mg2+, etc.), detergents or other buffer components to the appropriate final concentration when used. For rapid and complete polymerization, degas using a vacuum extractor until bubbles are not strongly produced. If a decontaminant is used, add it after degassing to a final volume of 200 ml of the gel mixture.
(3) Remove the piston of the syringe, seal the lower hole of the syringe with paraffin film and keep it upright, add 70.7 μl of 100X peptide storage solution (or buffer, used in the gel strip at a peptide concentration of 0), add 7 ml of gel mixture, 14 μl of TEMED, seal the end of the syringe firmly with your finger, gently push the piston back to the proper position of the syringe, invert the syringe so that the hole of the needle is upwards to allow the air bubbles to rise to the top, remove your finger and the paraffin membrane, do not point the syringe towards yourself as a small amount of gel mixture may be sprayed out, push the piston until it is completely inside the syringe and then block the needle hole again. Reverse the syringe several times to mix the gel mixture, remove your finger and push the piston to allow the gas to spill out of the syringe until the gel mixture is just about to spill out.
(4) Attach the 21 gage needle and slowly pour the gel through the top space of the device between the two glass plates until the gel reaches the line drawn between the first incision.
(5) Wait 10 min to allow the gel to polymerize, and while waiting, remove the syringe and discard the remaining mixture into an appropriate container. Wash the syringe and needle with distilled water for the next use, taking care to ensure that no water remains.
(6) When gel polymerization is complete, aspirate the upper layer of unpolymerized gel solution by sliding a piece of Whatman paper, just wide enough to be inserted between the glass plates, between the two plates.
(7) Repeat steps (4)~(6), sequentially varying the concentration of peptide in the gel to perfuse gel strips labeled 1~8.
(8) Reduce the gap between the bottom of the gel and spacer S and fill the last gel strip (labeled 9). Tilt the device to allow the gel solution to flow to the bottom of the filling comb and wait 10 min for gel polymerization.
(9) Lay the device flat as in Fig. 11.7 and raise it slightly (about 5 cm) at the end of the gel comb. Remove the gel comb and blot the remaining unpolymerized gel with Whatman paper. 
(10) Prepare 7 ml of gel mixture with a peptide concentration of 0. Add 14 μl of TEMED, then perfuse until the gel mixture reaches the edge of the top plate, add 1 ml of TEMED, insert the comb, and polymerize for 15 min.
(11) Remove the bottom spacer and comb, and load the filled gel into the electrophoresis tank.
(12) Add 0.5% TBE buffer (add salt or detergent) to the electrophoresis tank, wash the sample wells and the empty space after removing the bottom spacer without pre-electro-ice, and add the RNA samples to the appropriate sample wells, which generally can only contain 2 μl of sample.
(13) The gel should be electrophoresed at a constant low voltage to minimize heat generation. A 21 cm X 36 cm size, 0.8 mm thick gel can be separated well at 3 W (regardless of salt concentration), and the inches of electrophoresis should be controlled so that the RNA and peptides just do not run out of the gel.
(14) After electrophoresis, remove the gel device, remove one electrophoresis glass plate, cover the gel with a plastic flagon film, mark the peptide concentration of each lane on the plastic film, peel the gel from another plate, transfer it to the plastic film, and cover the gel with Whatman paper.
(15) Dry the gel in a gel dryer for 1 h. Stick on a two-point luminescent label as a reference, and expose the gel to the negative for an appropriate amount of time.
(16) Develop the negative, allow it to dry, align the gel to the negative using the luminescent labels, mark the interface between the peptide-containing region and the spiked-well region, and measure the distance from that point to the center of each strip, or to the bottom of the strip (when the strip is trailing or crosses the demarcation between the two lanes); it is important that consistent measurements be used in a single experiment.