Synthetic beta amyloid 1 42 is resuspended in HFIP to first modernize the peptide and later concentrated down to a film for ligamentization. The film is RESUSPENDED in DMSO, diluted in phosphate buffer and incubated overnight. Finally, the oligomer peptide is diluted with recording buffer and perfused onto acute hippocampal slices where the effective beta amyloid exposure on synaptic plasticity is assessed.
Welcome to the lab of Avio Orio at Columbia University in New York City. Hi, today we will show you a procedure we have optimized for the preparation of all Li Rise beta amyloid, which is based on a protocol originally proposed by Stein and colleagues in 2003. In this laboratory, we used this procedure to study amyloid beta induced synaptic dysfunction.
So let's get started. Allow the vial of Lyophilized beta amyloid powder to come to room temperature for 30 minutes to avoid condensation when it is opened under the fume hood. Open the vial and add ice cold HFIP to obtain a final beta amyloid concentration of one millimolar.
Close the vial and vortex to mix using a Hamilton syringe quickly divide the solution equally between three low binding polypropylene micro centrifuge tubes. Close the tubes and incubate for two hours at room temperature to allow the peptide to mize open the tubes and place them in a speed vac to concentrate the solution. Spin at 800 Gs under vacuum for about 15 minutes.
Make sure the temperature does not go above 25 degrees Celsius to avoid protein degradation. The spin is done when a clear dry peptide film is seen at the bottom of the tube. The film may be difficult to see seal the tubes and store unused aliquots at minus 80 degrees Celsius for up to six months in the hood at DMSO to resuspend the peptide film to a final concentration of five millimolar sonicate in a water bath for 10 minutes.
To ensure complete resus suspension of the peptide store 1.6 microliter aliquots of the five millimolar beta amyloid solution at minus 20 degrees Celsius. Each aliquot should be thawed only once the day before the experiment thaw one aliquot of the five millimolar beta amyloid solution and add 98.4 microliters of sterile phosphate buffer to a final volume of 100 microliters vortex for 30 seconds and incubate at four degrees Celsius for 12 hours. To allow for ligamentization to start an experiment, incubate fresh hippocampal lysis for 90 minutes.
In standard recording buffer, the stimulating electrode is placed on the shafer's collaterals and the recording electrode is placed on the stratum radium. Once a response has been found and the baseline is stable for 10 minutes, dilute the all liga beta amyloid to 200 nano molar. With recording buffer beta amyloid binds non-specifically to glass, so use plastic as much as possible.
Perfuse the beta amyloid solution onto the slices for 20 minutes. Deliver your preferred toan X stimulation. For example, a theta burst immediately after the tetanus switch back to profusion with normal recording buffer.
Monitor the recording for at least two hours post tetanus to confirm LTP induction and see the effects of beta amyloid treatment. We have just shown you how to prepare oligomeric beta amyloid one to 42 for the induction of synaptic plasticity impairment on hippocampus slices. When doing this procedure, it is important to remember that over drying the peptide film can lead to incomplete a ligamentization.
It is also critical to use plastics for beta amyloid solutions to prevent non-specific peptide binding. Thanks for watching and good luck with your experiments.
