Hello Changhua Shi,

To your question: How to keep the beta amyloid 42 to be monomer? Does anyone have any idea of how to keep the amyloid peptide as a monomer at neutral pH?

That has been a big question for a long of time, and the answer is not straightforward. In short, to my incomplete knowledge, there is no practical way to keep it as a monomer in concentrations that you can work with. Do not try to freeze aliquots because freeze/thawing leads to aggregation. In other words, one of your biggest concerns working with amyloid beta 42 is reproducibility to the experiment you are doing. For example to quantify for an ELISA is OK some solvents are not a big deal for the peptide antibody interaction, for effect on cells or interaction with membranes, lipid etc you want to be extra careful, the peptide does not behave as a "standard" peptide.

Generally most people get the amyloid beta 42 peptide as a synthetic peptide in a TFA salt form, TFA is used is used the elution  from the resin used for the synthesis. TFA is a strong acid that lowers the pH and strongly tend to aggregate -in various ways- this peptide. In other words, while you may buy a relatively pure or very pure peptide, it comes already aggregated and with several aggregation seeds, and as such it will continue to aggregate once you add a water base buffer.

At very low concentrations the aggregation is slower, also at lower temperatures (~4C). 

There has been various ways to try to "monomerise" the peptide before using for experiments.

1- One uses the solvent HFIP or HFP (hexafluoroisopropanol) which effectively does disaggregates amyloid beta 40 and 42, but you must make sure you fully eliminate even traces of the solvent because it binds to the peptide and is also cytotoxic. To do this once you have fully disaggregated the peptide, you can lyophylize already in aliquots ready to your uses. then you have not prevented aggregation but you always start from the same point (when you resuspend the powder), you can see it as time = 0. The procedure is time consuming and needs specialized equipment.

2- Another approach dissolves the peptide on NaOH or NH3OH at high pH, prior to your experiments, I do not believe it would be wise to make aliquots of these, but many people do use this approach because of its simplicity. Talking to Peptide Chemists some have suggested that you may damage some amino acid residues this way, however I would prefer some one else addresses that.

3- Another approach is not to treat the peptide and simply dissolve it and then to separate the various fractions by size exclusion chromatography. Monomers obtained by these approach can be preserved in ice for up to 24 hrs at a concentration of 10-12 microM or lower. However, even this approach has some degree of dimers and trimers, and is not stable. Again when you collect the right fraction start counting as time = 0.

I am sure there are other ways, so look out there for more.

Hope this is useful,

Ricardo Capone

Why do you need to dissolve it? Why not just lyophilized until use?

Does it need to be in aqueous solution? You can try DMSO. If aqueous, very dilute basic solutions may help, but I'd be it will still fibrillize with time. 

generally we lyophilized it until use , it's not stable in any solution！

the short chemist-answer: it is impossible to keep Abeta peptides as monomers in solution - at least in near-physiological conditions - 

Welcome to the world of "peptide from Hell"!

The only way to have monomeric AB42 if you order it from company is to dissolve in 0.1 M NaOH and run size-exclusion chromatography of not very concentrated peptide solution immediately before performing your experiments. You  take the monomeric fractions from gel-filtration and use them. Not shaking, no shearing force and low temperature will help you to keep it useful for a while (a couple of hours). There is no way to keep it monomeric at neutral pH. Even at high pH you will get a mixture of different species. If you want consistent results - use immediately.

The AB42 dissolved in NaOH can be frozen in liquid nitrogen and kept at -80 deg until use. Then you run gel-filtration every time you need monomeric AB42.

We have used recombinant AB42 expressed in E.coli in complex with Affibody protein and denatured the complex to get monomeric AB42. You can check for details in our papers:

http://www.ncbi.nlm.nih.gov/pubmed/18973685

http://www.ncbi.nlm.nih.gov/pubmed/20713699

Good luck!

maybe this paper can help you?

Article A standardized and biocompatible preparation of aggregate-fr...

Hello,

about your question there is a way to produce monomeric bata-amyloid 42: you should dissolve the lyophilized peptide  in Guanidine-HCl 6M and then you can use a Size exclusion chromatography (SEC) to be sure to eliminate all oligomers and intermediates that you have in lyophilized beta-amyloid 1-42.

Then, you should keep the solution a low concentration lower then 10uM and store for 1 week a 4C

I hope this answer could be useful for you

Best

Simona

Thanks for all of these help. I have seen some paper that use SEC column or a centrifuge tube with a 5 kD cut off membrane to separate the monomer from oligomer. For me these methods are all reasonable but after I got the monomer from these method are they stable or how long can they keep the monomer status in a neutral or basic pH such as 7.4- 8.0? Additionally, my peptide is order from Genescript which is a click peptide. The peptide can switch to nature status in  a basic buffer, so I need to covert the peptide first and then to prepare the monomer. Does anyone use the click peptide before for this kind of study?

Thanks  again for all of your help.

Steven

Dear Changhua Shi,

would you be so kind and send me a link to the paper where they used the 5 kDa cut-off membrane to separate the monomer from oligomer?

thank you 

Eduard