The overall goal of this procedure is to monitor and compare the aggregation kinetics of unmodified and hyper phosphorylated tau protein species. This is accomplished by first generating unmodified and hyper phosphorylated tau protein by the zippers assisted catalyst system. The second step is to set up the aggregation assays to be suitable for either of two different instruments.
The tau aggregation process is monitored by the fluorescence of two different thio. Flavin dies, theof flavin S and theof flavin T.The final step is to plot and compare the aggregation curves. Ultimately, the kinetic changes of thio flavin fluorescence from binding to aggregated TA species are used to show the hyper phosphorylation dependent enhancement of tau fibrillation in vitro.
Hi.Welcome to our JOV article. In vitro aggregation assays using hyper phospho tau protein. My name is Minko, and the experiments will be conducted and demonstrated byway.
And For this protocol, a reasonably fresh aggregation buffer made within the last month. Just before its use add DIO three etol to one millimolar also required A filtered Theo Flavin tea or S stock solution. Hein stock solution, both storable at minus 20 degrees Celsius and towel protein stored at minus 80 degrees Celsius.
Pull the towel on ice, then adjust its concentration using the aggregation buffer as required for consistency. Spin down the towel with aggregation buffer and 20, 800 G for 10 minutes of four degrees Celsius. Now transfer the supinate to another tube and keep it on ice until it is time to assemble the aggregation reaction.
Set up the aggregation mixture in 1.5 milliliter micro centri tubes. According to the table, 100 microliters is enough for one aliquot of reactants. Calculate the total volume based on the number of reactions and increase it by 10%to account for pipetting errors.
Pepper in can be replaced by raonic acid or aggregation buffer if needed. Mix the reactions by inversion and transfer them to 37 degrees Celsius without vegetation. After every 24 hours, replenish the reactions with a supplemental one millimolar of DTT to ensure a reducing environment to measure fluorescence.
Warm up the spectra fluorimeter for about 10 minutes. This will improve the consistency of the measurements in the computer software. Select the real-time display mode found in the instrument control center.
Set the excitation wavelength to 450 nanometers with the slit at two nanometers and set the emission wavelength to 510 nanometers with the slit of five nanometers. Then return to the instrument control center and choose constant wavelength analysis. Press the add key in the upper frame to add a set of wavelengths.
Set the acquisition parameters of the standard error to one and the maximum trials to three. Then click add. Next click go to open the data display window.
Now in the data display window, click on start act to open the new sample dialogue box and choose unknown for sample type. Then prepare the reactions to take measurements to every 100 microliters at 98. Microliters of aggregation buffer, and two microliters of three millimolar theof flavin S or T pipette the mixture several times to ensure good mixing.
Transfer all 200 microliters of one mixture to an FA three qve and load it into position. Then lick run to collect the fluorescence data After removing the solution from the vete, rinse it three times with distilled water and dry it in an airstream. This assay includes the fluorescent dye in the aion reaction, which allows continuous measurements and is better done with an automatic mat well plate reader.
A regular for parameter also works is limited by the speed of manual operation. Set up the aggregation mix in a 96 well opaque black plate. With 360 microliter a well volumes 200 microliters is enough for each time point measurement.
Mix each reaction well with pipetting. Incubate the 96 well plate at 37 degrees Celsius and every 24 hours add a supplemental one milli molar of DTT to ensure a reducing environment at each time point. For a measurement, turn on the multi-mode microplate reader and the computer at least 10 minutes in advance in the computer software.
Set the temperature to 37 degrees Celsius and select the fluorescence intensity mode. Then set the excitation wavelength to 450 nanometers and the emission wavelength to five and 10 nanometers. Insert the 96 well plate into the draw and press the read key.
After taking measurements, immediately return the plate to the incubator before analyzing the data, the continual mode assay can be performed using a compact spectrophotometer for this assay. Set up the aggregation mixture in 1.5 milliliter micro centrifuge tubes. 200 microliter reactions are enough to measure a time point.
Invert the tubes to mix. Set up the reactions at 37 degrees Celsius or room temperature and add a supplemental one millimolar of DTT every 24 hours to take measurements. Set up the urometer and software as done for the no die terminal assay.
Transfer the entire mixture to a vet and position it into the sample holder in the sample compartment and close the lid. Click run to collect the fluorescence data at the appropriate time intervals. If this interval is short, like 30 or 60 seconds, it is okay to leave the Q vet in the machine between measurements until the last time point is reached.
Return the reaction from the vete to its sample tube and return the tube to the incubator When completed. Lean the vete as previously instructed using recombinant tau and P tal. Two different protocols were established to compare the kinetics of aggregation of tau and p tal, taking advantage of the strong fluorescent emission of theof flavin t and s upon binding to amyloid protein aggregates, including tau and p tal, with or without the fluorescent dye.
In the aggregation reaction, there was consistent enhancement of tau aggregation by hyper phosphorylation. Typically, T and p tal liga rise quickly within the first 30 minutes before a significance slowdown, including theof flavin. T in the aggregation reactions cause significant retardation in the rate of aggregation.
Both Theof Flavian s and t approached a plateau after reacting for 160 minutes. Bio flavin s, on the other hand, did not cause appreciable slowdown of aggregation. Following this procedure, additional central nervous system to isoforms or other aggregation prone proteins in neurodegeneration, likein can be performed using the assay in order to understand the contribution of protein ization in pathogenesis After its development.
This technique of preparing an hyper phosphate top protein PA way for research in the field, numerous disease to explore our therapeutics and drug development.
