Hi There,
I need to prepare oligomeric amyloid beta for injection into the rodents and Ive seen HFIP is used as amyloid beta solvent in many papers as well as my current protocol. I was wondering to know if there is any appropriate substitute instead of HFIP, since this chemical is highly toxic and corrosive?
Thanks in advance.
Sara
An option is to dissolve amyloid beta in a very small amount of DMSO (to 5 mM amyloid beta) and then dilute to your favorite concentration the peptide with your solvent of choice (i.e. PBS) before injection.
Depending on the "waiting" times you might have Abeta oligomers.
A standard way used in the past to prepare Ab oligomers is the preparation known as ADDLs (https://www.ncbi.nlm.nih.gov/pubmed/?term=J+Neurochem+2001+MP+Lambert).
A disadvantage of DMSO is that it is an oxidizer and a fraction of your peptide might be oxidized (in Methionine 35) if staying in solution for a long time.
Whatever you do you can not be sure that your amyloid will be beta after injection.If you want to interpret
your in vivo findings structurally you should be very cautious.
Dikeos Mario
The reason HFIP is used is to ensure that the peptide is entirely monomeric before starting aggregation experiments. I think most protocols will monomerize with HFIP at some point, as it has become the standard. We use a process of sonication combined with HFIP and filtration to remove as many non-monomer species as possible.
However, if your goal is to not inject any HFIP into your rodents, then this protocol by Lee et al. might be useful.
Article Amyloid-?? Forms Fibrils by Nucleated Conformational Convers...
That being said, it is quite difficult to characterize oligomeric species, and I agree with Dikeos Mario Soumpasis in that caution should be taken when interpreting your results.
Best of luck,
Mat
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