The overall goal of this procedure is to generate and purify good quality and nool pyrophosphate. This is accomplished by first using IP six as a substrate for the IP six kinase, one enzyme to generate IP seven. Next, the enzymatic reactions are loaded onto a polyamide gel and the band corresponding to IP seven is excised.
The IP seven is then isolated by subsequent dehydration hydration cycles. The final step of the procedure is to confirm the purity of the product and to determine the concentration. Ultimately, results can be obtained that show the biological activity of IP seven through incubation of IP seven.
With VIP one kinase and with the IP seven phosphatase DDP one, these reactions generate IP eight and IP six respectively. Do this Met can provide insight into IP seven signaling. It can also be applied to other system, such as in part of phosphates like IP eight or the IP five derived PPIP four.
This protocol begins with the preparation of enzymatic reactions in which IP six kinase one or VIP one convert IP six to the pyro phospho related isoforms. For this demonstration, histamine tagged IP six, kinase one and GST tagged VIP one enzymes were purified from e coli according to a previously described procedure. To begin, prepare 10 to 20 independent enzymatic reactions containing IP six and his IP six kinase one or GST VIP one as described in the written protocol.
Adjust the volume to 50 microliters with milli Q double distilled water. After briefly spinning the tubes, incubate the reactions at 37 degrees Celsius overnight with rotation. Prepare the poly acrylamide gel apparatus using 24 centimeter long, 18 centimeter wide glass plates, 1.5 millimeter wide spaces and a 16 lane comb.
Next, pour a previously prepared gel solution between the pre casted glass plates at a volume of 50 milliliters per gel. Insert the comb and let the gel polymerize for 30 to 60 minutes at room temperature. Once the gel has polymerized, transfer the apparatus to the cold room.
Pre-run the gel in one times TBE for about 30 to 60 minutes at 200 to 300 volts, meanwhile at one times orange G dye. To each reaction, prepare a standard control sample containing two mol of IP six. Following pre-run, wash each well thoroughly with running buffer.
Using a syringe attached to a 21 gauge needle to remove any precipitate. Then load the samples into the gel, avoiding loading the first two and last two lanes of the gel. Finally, run the gel overnight at seven milliamps per gel until the orange G dye Band is within the last 10 centimeters of the bottom of the gel.
To begin isolation of IP seven, disassemble the gel apparatus and carefully remove one glass plate leaving the gel on the other one. Cut a small portion of the gel from just above the orange GD band to the bottom, including the IP six standard and one sample lane. Stain the cut portion of the gel with toluidine blue for a few minutes until the Anatol Pyrophosphate band appears.
Meanwhile, place the previously removed glass plate back on top of the gel in order to prevent the unstained gel from drying. Transfer the stained portion of the gel to a DES staining solution for a few minutes in order to wash away any excess of toluidine blue, then reposition the gel with the unstained gel. The IP seven band should be visible since it runs slightly slower than the IP six standard A TP, which runs faster than IP six should also be visible.
Next, use a razor blade to cut the IP seven band on the unstained portion of the gel. Exploit the IP seven migrating position determined with the stain gel. To estimate the location of the IP seven band, put the IP seven band that was cut from the gel into a 15 milliliter tube and add 10 milliliters of milli Q double distilled water.
Rotate the tubes for 10 minutes at room temperature following incubation, discarded the liquid by decanting to remove excess TBE and microscopic acrylamide particles. Subsequently perform two dehydration hydration cycles by first adding five milliliters of 50%methanol to the tube containing the IP seven gel. Rotate the sample at room temperature for two hours.
Then transfer the gel slice to a new 15 milliliter tube containing five milliliters of milli Q double distilled water and rotate at room temperature for two hours. Keep the methanol containing tube for further use following incubation. Repeat the dehydration hydration cycle once more to concentrate the eluted IP seven.
Combine the previously added five milliliters of water and five milliliters of 50%Methanol. Dry the combined solution using a speed vac heated at 60 degrees Celsius. Once the samples are nearly dry, transfer the remaining liquid to a 1.5 milliliter centrifuge tube.
Spin the tube for two minutes of 5, 000 rotations per minute. Collect the SUP natant leaving the bottom 20 to 30 microliters since it may contain a acrylamide particles. Transfer the sample into a fresh 1.5 milliliter centrifuge tube.
If necessary, continue the drying process using an unheated speed vac. The recovery of IP seven is dramatically reduced if the samples dry completely. Therefore, it is imperative to terminate the drying process when the samples reach the volume of 100 to 300 microliters.
Once the isolated IP seven has been recovered, load two to five microliters of the sample onto a page gel to determine its concentration and purity. Load several dilution of IP six as concentration standards and four mol of polyp marker. After running the gel, visualize the inital pyrophosphate isoforms by staining and des staining the entire gel with toluidine blue solution.
Following the procedure just described following toluidine staining, the concentrations can be determined by scanning the gel and comparing the differences in intensity between IP six and IP seven. Using imaging software such as Image J, the preparative enzymatic conversion of IP six to IP seven. Using IP six kinase one and VIP one enzymes can be easily resolved using page analysis.
The loading of IP six as a size control together with toluidine blue gel staining allows for the identification of the pyro phosphorylated derivatives. Since they run slower, depending on the number of phosphate groups present on the inital, the procedure described above resulted in the easy purification of IP seven. Furthermore, analysis of the purified anatol pyrophosphate by page revealed the purity of the IP seven.
Interestingly, the one three pyrophosphate IP five isomer of the IP seven product of VIP one migrate slightly slower than the five pyrophosphate IP five isomer of VIP seven that is generated by IP six kinase one. The use of IP six standards permits an easy quantification of the concentration of the purified IP seven before using IP seven. For further experiments, its biological activity can be assessed.Five.
Pyrophosphate IP five is incubated with FIP one and with the IP seven phosphatase DDP one routinely. The purified IP seven is converted to IP eight by VIP one and to IP six by DDP one. Following this procedure, other methods like a prolonged tolin in blue staining can be performed in order to answer additional questions like isolation of IP eight or other phosphorylated in isoforms forms.
