Bachem suggests reconstituting the peptide in ammonia or 1mM NaOH to revert aggregation. This sounds like a good idea so the entire batch does not immediately clump together. I was planning to reconstitute in 1mM NaOH. However, I have heard that you can incubate these peptides from room temp. to 37C or shake them to make the aggregates form. I am planning to treat cells with this for cytotoxicity assays. Does anyone have suggestions or experience with using these peptides?
I would have a look into this publication: Globular amyloid β-peptide1−42 oligomer − a homogenous and stable neuropathological protein in Alzheimer's disease by Barghorn et al. They describe how to get stable oligomers and show cytotoxicity.
Thank you Claudia. I found another one with some similar methods. -In Vitro Characterization of Conditions for Amyloid-β Peptide Oligomerization and Fibrillogenesis*-WB Stine. These are both helpful.
Thank you Regis! This is similar to protocols that I am seeing in papers. The one reason that I have not resuspended the peptide in DMSO, however, is because when reconstituting this peptide at 1mg/ml, it is at a concentration of just above 200uM. That means that when diluting it out further with culture medium, a huge part will still be DMSO. I am using the peptide for cytotoxicity experiments, and I'm sure that amount of DMSO will be toxic to the cells, and I will see toxicity in the vehicle controls as well. Are you able to dissolve this peptide in DMSO at a concentration higher than 1mg/ml? This is an expensive peptide and I'm also concerned with losing a lot of protein by adding HFIP and then evaporating it. Do you not lose much when drying it with nitrogen gas? Thank you for all the tips!
Thank you very much. This now makes much more sense that the HFIP step is to increase the solubility in the DMSO so you are not having to use as much DMSO. I will plan to use this protocol if I can just find some less expensive amyloid beta to work with! Best~Mandi
Regis- I hope things are going well. I have one more questions on the use of this HFIP for handling amyloid beta when you get a chance. Apparently, HFIP is fairly hazardous and the MSDS says that it may require respirators. Do you use a respirator when working with this or just work under a fume hood? Is it okay to let it evaporate under the hood? Thank you for all of your help.
Mandi
Hello everybody,
I was wondering if anyone used the protocol in "In Vitro Characterization of Conditions for Amyloid-β Peptide Oligomerization and Fibrillogenesis" -WB Stine. I am also interested in using the conditions for the formation of oligomeric amyloid beta but I cannot find in Europe where to buy Ham´s F12 w/o phenol red. Is there any other media that could be used?
Thank you!
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