Recombinant alpha, beta, and gamma synucleins stimulate protein phosphatase 2A catalytic subunit activity in cell free assays. Hello, my name is Sovanarak Lek. I am a medical student at Texas Tech University Health Sciences Center, El Paso, Paul L.Foster School of Medicine.
In this video, I will be demonstrating a protein phosphatase two way color metric assay using recombinant synuclein proteins. Preparation of buffers and radiants. Preparing p-Nitrophenol Phosphate Buffer.
To prepare 50 mils of of pNPP buffer dissolve Trizma base in 25 mils ultrapure deionized water in a beaker. Add a clean stire bar and place on magnetic stirrer. Once Trizma dissolves, add calcium chloride with continued stirring.
Adjust pH to 7.0. Transfer the solution to a graduated cylinder. Bring the final total volume to 50 mils with ultrapure deionized water.
Preparing malachite green solution. Malachite green solution requires three solutions, parts A, B, and C.Solution A is four molar hydrochloric acid. Solution is a combination of ammonium molybdate plus solution A.Solution C is 0.045%malachite green oxalate salt.
To prepare solution D, mix one part B to three parts C, one to three ratio. Stir one hour at room temperature. Finally, filter solution D through a 22 micrums membrane and store at four degrees Celsius.
Protect from light. Preparation of activator. Activator is one percent Tween 20 solution.
Pippette 10 microliters of Tween 20 in 990 microliters of ultrapure deionized water. Vortex until Tween 20 dissolves completely. Preparing malachite green plus Tween 20 working solution.
Add activator to solution D in a ration of one to a 100. Preparing threonine phosphopeptide. To prepare a two mM pT stock solution, add 550 microliters of ultrapure deionized water to one milligram pT.
Vortex until phosphopeptide is completely dissolved. Place on ice to use immediately. Store extra pT aliquots at minus 20 degrees Celsius.
Synuclein preparation. Resuspend one milligram of synuclein in mil ultrapure deionized water at a final concentration of one milligram per mil. Vortex gently to dissolve.
Use immediately and store unused aliquots at minus 80 degrees Celsius. Preparing phosphate standards. Phosphate standards step one.
To prepare 1 mM phosphate stock solution dissolve potassium phosphate in ultrapure deionized water in a beaker with a stir bar. Transfer to graduation cylinder and bring the final volume to 100 mils. This is a 10 mM solution.
Filter the entire solution through a 22 micros filter using a large syringe. Dilute this to 1 to 100 to make 1 mM phosphate solution to be used for assay standards. Phosphate standards step two.
Add the appropriate amount of phosphate plus ultrapure deionized water to 1.5 mils tubes as per table. This will yield tubes of 150, 300, 600, 1, 200, and 2, 400 pmol phosphate. And store aliquots at minus 20 degrees Celsius.
PP2A activity assay. Prepare PP2A assay in 1.5 mil tubes on ice. Add 43 microliters pNPP buffer.
Add one microliter of human recombinant PP2Ac. Add synuclein and incubate PP2A plus synuclein 30 minutes on a Nutator at four degrees Celsius. Next, add 16 microliters of 2mM pT.
Incubate 10 minutes at 30 degrees Celsius in water bath. On a flat bottom 96-well plate, add 25 microliters of each phosphate standards in duplicate from low to high. Pipet 24 microliters of each PP2A assay in duplicate onto the same 96 well plate with the standards.
Pipet 75 microliters of solution D containing Tween 20 to each sample and standard. Upon addition of MGT, you will see a color change in wells when free phosphates are present. Photometric analysis.
Analyze on a spectrophotometer set to 630 nanometers. Representative results. Here we have a representative PP2A assay and the graph demonstrating the standard curve.
On the left are the phosphate standards. The more phosphate that the wells contain, the darker the shade of green. One the right we have PP2A that was incubated with alpha, beta, or gamma synucleins at different molar concentrations.
Higher concentrations were more effective at stimulating PP2A catalytic subunit activity. Here we see the graphical representation of PP2A activity in response to varying concentrations of alpha synuclein, beta synuclein, and gamma synuclein. Alpha synuclein, beta synuclein, and gamma synuclein were all significant stimulators of PP2A activity at concentration of one to two micromolar.
Only alpha synuclein and beta synuclein were significant stimulators of PP2A activity at 0.5 micromolar concentration. Figure three shows the interaction of PP2Ac with endogenous synucleins in mouse brain. In A, recombinant alpha, beta, and gamma synucleins are shown as positive controls.
In B, co-immunoprecipitation was done using the PP2Ac-specific antibody 1D6, which brought down PP2Ac and also brought down considerable interacting alpha synuclein but almost no beta synuclein. No gamma synuclein came down with PP2Ac in the co-immunoprecipitation. This co-immunoprecipitation data suggests that alpha synuclein is the endogenous modulator of PP2Ac in vivo.
We want to thank everyone who helped us in making this video. So, in conclusion this is Ruth Perez. Sonavarak Lek.
Javier Vargas. Wishing you great success. Goodbye and good luck.
