This method can help answer key questions in the neuropharmacological field. The main advantage of this technique is that it's simple, reliable, and reproducible to form beta-amyloid oligomer. We show demonstration of this method is critical as steps for preparing beta oligomer are difficult to learn.
First, adjust the concentration of A-beta monomer solution to 0.25 milligrams per milliliter by adding 900 microliters of double-distilled water to the tube. Then continue evaporating the monomer solution with high purity nitrogen gas until the volume reaches about 850 microliters with a concentration of about 0.29 milligrams per milliliter. The presence of HR5P affects the formation of A-beta oligomer.
Therefore, evaporating as much as possible is a critical step of this procedure. Next, use double-distilled water to dilute the curcumin stock solution to prepare a working solution of 0.2 and two micromolar. Maintain a ratio of one-to-one and mix the A-beta solution with curcumin to achieve the final concentration of curcumin at 0.1 and one micromolar.
Then, leave the solution on a magnetic agitator. Attach a plastic divider box to the magnetic agitator. Position two magnetic stir bars at the two corners of the box, then place the tubes with the samples in the center of the box.
Continue shaking the box at room temperature for 48 hours at 60 revolutions per minute, then centrifuge the tube at 18, 000 times G for 15 minutes at four degrees celsius. After centrifugation, collect the supernatant. For dot blotting analysis, take a nitrocellulose membrane and cut it into one-centimeter wide strips, then place two microliters of the sample evenly on the strips one and two with each point interval at 0.5 centimeters.
When squirting the sample on membrane, it's necessary to spread evenly and the different droplets are controlled so that the different samples are not linked together. Next, incubate the strips at room temperature for five minutes for the droplets to dry out. After five minutes, incubate the strips with 5%bovine serum albumin for 30 minutes at room temperature.
Once the incubation is over, wash the strips with TBST buffer for five minutes. Aspirate the buffer after the wash is done, then incubate strip one with anti-oligomer antibody A11 and strip two with anti-A-beta antibody 6E10 solution for an hour. After one hour, wash the strips with TBST for five minutes each three times, then aspirate the buffer and incubate the strips with secondary antibody solution for 40 minutes.
After the secondary antibody incubation, again wash the strips with TBST for five minutes each three times. Then, add about 300 microliters of mixed ECL fluid to the surface of the moist strips, then expose the strips in an automatic chemiluminescence imaging system. TEM studies show the absence of any visible aggregates in the HFIP-dissolved A-beta monomer solution, however after 48 hours of incubation, the same monomer form oligomers, which are mainly globular aggregates with a diameter of 10 to 80 nanometers.
On subjecting the A-beta monomer and oligomer-rich samples to dot blot analysis, it shows the presence of A11 positive A-beta oligomers in A-beta oligomer-rich samples. Interestingly, A-beta 6E10 positive A-beta peptides are present in both the A-beta monomer and oligomer-rich samples. Next, semi-quantitative analysis is also done to express the specific antibody-positive A-beta monomer and oligomer-rich samples as a percentage of control.
A11 positive A-beta peptides are significantly higher in A-beta oligomer-rich samples in comparison to the monomers. On the contrary, A-beta 6E10 positive A-beta peptide is present in both the monomer and oligomer-rich samples. In fact, A-beta oligomers are also co-incubated with curcumin and A-beta oligomer inhibitor.
Curcumin leads to significant reduction in the relative amount of A11 positive A-beta oligomer with decreased A11 staining in the curcumin co-incubated A-beta oligomer samples. However, varying concentrations of curcumin do not affect the number of A-beta peptides in the 6E10 positive monomer and oligomer-rich samples when compared to A11 positive samples. Once mastered, this technique can be done in several hours if it performed properly.
After its development, this technique paved the way for researchers to explore drug candidates for neurodegenerative disorders such as Alzheimer's Disease. After watching this video, you should have a good understanding of how to prepare A-beta oligomer and to evaluate its amount.
