Amyloid-Beta separations

I’ve been working recently with amyloid-beta (AB) provided to me by some people in Brad Nilsson’s lab.  These experiments are related to the HMM diffusion experiments for our collaboration with Harold Smith.  Basically, AB fibrils are very long (microns) and we are looking for ways to either prevent their aggregation from smaller peptides/oligos, or to look for compounds that break up these large fibrils into smaller pieces.  pnc-Si would provide the material to separate these different size populations.

In this experiment, I was provided some AB40 fibrils and AB40 oligos (non-specific peptide aggregates ~1nm in size) for a diffusion test.  To set this up, I put 40uL of sample on 1 side of SC358 in a round Sepcon and 40uL of buffer (PBS for oligos and serum-free, but with phenol red, DMEM for fibrils) on the other side of pnc-Si.  I placed the samples in 15mL conicals to reduce evaporation and let them site for 24 hours at room temp (in our lab ~20C).  I then performed the Quant-IT assay. Below are the data for an AB40 oligos vs. fibrils separation with out pnc-Si membranes.  Since I only had 1 standard curve, I left the y-axis in relative fluorescence units (fluorescence from the Quant-IT assay).

I wasn’t sure if the Quant-IT would detect this samples since they aren’t ‘regular’ proteins, but it seemed to do fine. The standard curve for AB40 fibrils is slightly parabolic but other standard curves I’ve run weren’t linear, either.  I didn’t have enough AB40 oligo sample to run a standard. Both the fibrils and oligos went through these membranes.  However, the oligos were ~60% of the equilibirum concentration but the fibrils were only at ~ 36% expected equilibrium.  So, the fibrils were hindered a bit, although I didn’t really expected anything to make it through.  I’m trying to find out if there is smaller stuff in the fibrils sample.  As a first run, this separation seemed pretty good.