I am using HFIP to resuspend amyloid beta peptides in order to get amyloid oligomers, following the protocol from prof. Klein. However, HFIP is quite toxic for the cells, so after monomerization all the HFIP must be dry out.
The protocol says to leave the tube under the fume hood over night and then, since HFIP is quite hard to get rid of, to speed dry the tubes for 10 minutes the morning after.
My question is: if I want to avoid the over night step, can I skip it and let the tube in the the speed dry for a few hours instead? If so, how many hours are required to totally dry the HFIP? Will this affect the state of the peptides?
Many thanks.
I think storage of a-beta in HFIP for more than 24 hr is a way to guarantee complete monomerization, cause' I found that HFIP/TFE treatment time is crucial to the aggregation states in all my biophysical assays. You may use lyophlizer or any vacuum pump to speed dry with fixed/routine time, like 6 hours, then dissolve in buffer immediately for following assays you desired. Drying in hood may lead to contamination (especially for cell experiments)
Ppl who do in vitro experiments sometimes left trace of TFE/HFIP or even TFA to make it easier while handling highly aggregation-prone peptides, by not using vacuum pump to completely remove HFIP. But I think completely removing HFIP is ok in the case of a-beta. For cell experiments, personally i dont like trace of HFIP messing up.
*TFE = trifluoroethanol; TFA = trifluoroacetic aicd
I agree with Dr. Po-Wen Chen. Vacuum is probably the most suitable way to get rid of HFIP. You can also try nitrogen air flow for few hours.
Thank you both for your precious help. For how long would you advice to leave the solution on the vacuum in order to get rid of all the HFIP? For example, would 1h be enough?
For cell I think 6-12 hrs, to alleviate all possible toxicity from HFIP.
Praveen Kumar S E Here is the protocol I use (look at the link in the first comment): https://www.researchgate.net/post/Western_Blot_protocol_for_the_visualisation_of_the_Amyloid_beta_different_fractions
Hi all,
I am using HFIP to resuspend amyloid-beta peptides in order to get amyloid oligomers. Most of the protocols say to leave the tube under the fume hood overnight and then, since HFIP is quite hard to get rid of, speed dry the tubes for 10 minutes the morning after.
I was wondering, by overnight, how many hours do you mean? Can we let the HFIP dry out over 48 hours instead of overnight?
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