This method can answer key questions about the phosphorylation of the five-prime end of DNA or RNA molecules by a special class of enzymes known as polynucleotide kinase. The main advantage of this technique is it has the resolution to detect very small changes in DNA and RNA substrates. Begin by preparing the RNA enzyme kinase reactions.
For each reaction, combine one microliter of 500 nanomolar RNA substrate. 8.3 microliters of 130 nanomolar last one, GRC three and 0.2 microliters of five millimolar EDTA. Set the heat block to 37 degrees Celsius.
Then mix 0.5 microliters of an ATP sub stock from the concentration series with one RNA enzyme mixture and place the reaction on the heat block. Continue mixing the reactions and placing them on the heat block at ten second intervals. After a 60 minute incubation on the heat block, quench each reaction by spiking it with 10 microliters of urea loading dye.
Immediately perform downstream analysis or store the reactions at negative 20 degrees Celsius to be analyzed at a later date. To prepare a 15%denaturing acrylamide gel solution, combine 22.5 milliliters of pre-mixed 40%29 to one acrylamide/bis acrylamide solution. Six milliliters of ten x TBE.
28.8 grams of urea. And RNase free water to a total volume of 59 milliliters. Then gently stir the solution.
Heat the solution in the microwave for 20 seconds. Stir it and immediately return it to the microwave for another 20 seconds. Gently stir the solution until the urea is completely dissolved.
Place the glass beaker into a shallow water bath, containing cold water for five minutes, making sure that the level of cold water surrounding the glass beaker is above the level of solution inside the glass beaker. When the solution is cool, filter and de-gas it with a 0.2 two micrometer disposable filtration unit to remove particulates and microscopic air bubbles. Wash a short and long glass plate with soap and water then spray each plate with 95%ethanol and wipe the glass to remove any moisture.
Elevate the long plate off the bench top by placing it on top of a box. Then position 0.4 millimeter spacers along the long edges of the plate. Lay the short plate on top of the long plate, making sure that the edges of the short plate, long plate and spacers are aligned.
Then clamp each side with three evenly spaced metal clamps. Add 24 microliters of TEMED to the acrylamide solution and mix it. Then add 600 microliters of 10%APS and immediately pour the solution between the glass plates.
Pouring the acrylamide solution between the glass plate sandwich can be really challenging. To avoid air bubbles, tap the glass plate sandwich while you're pouring your solution. Carefully add a clean 32-well comb to the top of the glass plate sandwich.
And allow the acrylamide to polymerize for 30 minutes. To run the gel, set the heat block to 75 degrees Celsius. Remove the metal clamps.
And thoroughly wash and dry the glass plate sandwich. Position the plate sandwich and the gel apparatus with the short plate facing forward and prepare 0.5 X TBE running buffer by combining 100 milliliters of 10 X TBE with 1.9 liters of RNase free water. Add 600 milliliters of the running buffer to the upper and lower chambers of the apparatus.
Gently remove the comb from the gel and thoroughly rinse the wells with a syringe. Pre-run the gel at 50 Watts for 30 minutes. Then rinse the wells again.
Post spin the quench reactions and incubate them at 75 degrees Celsius for three minutes. Repeat the pulse spin and immediately load 10 microliters of each sample onto the gel. Then run the gel for three hours at 50 Watts.
When the run has finished, turn off the power supply and drain the upper chamber of the apparatus. Wash and dry the outer side of the glass plate sandwich. Then cover it with foil and transfer it to a laser scanner for imaging.
Mount the glass plate sandwich onto the stage of the laser scanner. Set the excitation and emission wavelengths for the desired floor for and image the gel. Shown here is a representative successful denaturing gel of a titration of ATP with a fixed amount of last one GRC three complex.
Addition of enzyme resulted in last one mediated RNA cleavage of the Saccharomyces cerevisiae ITS two RNA substrate, leading to a defined RNA fragment. Upon the addition of ATP, the C2 RNA fragment was phosphorylated by GrC3 PNK. To visualize the phosphorylation of the C2 RNA fragment, the relative amount of unphosphorylated and phosphorylated C2 RNA was plotted against the ATP concentration.
Our representative unsuccessful denaturing gel is shown here. The 21 nucleotide RNA substrate contained degradation products, which overlapped with the phosphorylated product and made it impossible to accurately quantify phosphorylation. In contrast, the shortest RNA degradation product could be successfully analyzed because this area of the gel did not contain any additional RNA species that hindered accurate quantification of its phosphorylated counterpart.
Most important thing to remember when attempting this protocol is to rinse the wells of the gel to ensure even loading of your sample. Following this procedure, an RNA turnover experiment could be performed. To measure rates of RNA degradation, phosphorylation often signals initiation of RNA degradation.
This technique paves the way for answering detailed questions about the activities specificity and kinetics of polynucleotide kinases.
