Bachem offers a broad range of amyloid peptides. Some of these peptides are notorious for their propensity to form scarcely soluble aggregates, which makes reconstitution difficult. As to help our customers, we have compiled a selection of literature references containing protocols for dissolving the peptides. We hope you will find some information, which could be helpful for your experimental work with our peptides.
Alzheimer`s disease (AD) is a neurodegenerative disease, characterized by extracellular amyloid β-peptides plaques in the brain. The amyloid β-protein consists of 39-43 amino acid residues, derived from proteolysis of a larger β-amyloid precursor protein (β-APP). Aggregation (or aging) of the amyloid β-protein is a requirement for its neurotoxicity . During this aging process, the amyloid β-protein undergoes conformational conversions, from soluble, monomeric random coil or α-helix conformation into aggregated β-sheet structures [2, 3, 4]. Several studies demonstrated that this conformational change within the aggregation process was affected by a number of factors, such as:
- the length of the peptide 
- solvent hydrophobicity 
- ionic strength [7, 8]
- pH-value [4, 9, 10]
- peptide concentration 
- initial aggregation state [12, 13]
- buffer type 
- counterions (e.g. CF3COO-vs Cl-) or other ions present [15, 16]
- the presence of partially oxidized or pre-aggregated forms (seeds) of the peptide [5, 8].
Considering this, it is not astonishing that even the smallest modifications could drastically change the aggregation rate, respectively the solubility.
HFIP-treatment of amyloid β-peptides
Preformed aggregates of amyloid beta-proteins can inhibit the solubility of the lyophilizate. The standard method to disassemble these aggregates is the treatment with 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). HFIP is a volatile solvent, which disrupts amyloid beta fibrils and generates monomers . Removal of the solvent in vacuo leaves behind a film of monomers, which can be reconstituted in DMSO and afterwards diluted with various solvents. HFIP treated amyloid β-proteins (1-40) (H-7438), (1-42) (H-7442) as well as (42-1) (H-8388) are available in the Bachem catalog.
Solubilization and aggregation of lyophilized amyloid peptides
In the following section you will find some examples from literature which describe the aggregation and solubilization of amyloid β-protein fragments.
For the solubilization of amyloid β-protein (1-42) (product number H-1368) Bachem recommends the use of a 0.1 M aqueous ammonia solution (1 mg/ml, pH > 9). The addition of ammonium hydroxide has been reported to assist the formation of monomeric amyloid β-protein .
J. Busciglio et al. dissolved the lyophilized amyloid β-protein (1-40) (H-1194) to a stock concentration of 1.0 mg/ml in either deionized water, 35 % acetonitrile / 0.1 % TFA or 100 % DMSO and then diluted it (1 : 100) directly into tissue culture medium. Lyophilized amyloid β-protein (1-40) was poorly soluble in saltcontaining buffers (e.g. PBS), but could be introduced into saline-containing solutions after being initially dissolved in one of the three vehicles described above .
In the article of G. Perini et al. amyloid β-protein (25-35) (H-1192) was dissolved at 1.5 mM in PBS, amyloid β-protein (1-40) (H-1194) at 1.5 mM in double-distilled water and subsequently diluted at 250 µM in PBS, and amyloid β-protein (1- 42) (H-1368) at 500 µM in double-distilled water. Furthermore, the authors reported that fibrillogenesis by amyloid β-protein (25-35) took place within minutes at room temperature, whereas amyloid β-protein (1-40) and amyloid β-protein (1-42) required 5-6 d at 37 °C. No fibril formation could be observed for amyloid β-protein (35-25) which was dissolved in the same solvent as amyloid β-protein (25-35). When the above mentioned amyloid β-proteins were dissolved in DMSO, they didn`t form fibrils and remained in solution . Contrary to the TFA salt (H-1194), the HCl salt of amyloid β-protein (1-40) (H-5568) was able to form β-structures in PBS within a few hours at 25 °C . Stine et al. describe the preparation of a variety of different aggregation states of amyloid β-protein (1-42) from a 5mM stock solution of HFIP-treated amyloid β-protein (1-42) in dry DMSO, which was vortexed and sonicated before use .
Faucher et al. describe the use of HFIP-treated amyloid β-protein dissolved in a solution, which does not contain DMSO. To achieve this, the HFIP-treated amyloid β-protein (1-42) was reconstituted in DMSO and afterwards eluted from a desalting column (dextran-epichlorohydrin copolymer) using an eluent containing Tris50-buffer and EDTA .
After treatment with HFIP, Tiiman et al. dissolved amyloid β-protein (1-42) in freshly filtered ultrapure water containing 0.02% ammonia at a concentration of 10-20 μM. After an incubation time of 5 minutes, this solution was further diluted and used for experiments .
In the study of C.W. Cotman and coworkers small aliquots (1 mg or less) of amyloid β-proteins were solubilized to a concentration of 250 μM with double-deionized water. Since aggregate formation occurred over time, immediate use of newly solubilized amyloid β-protein solutions circumvented the aggregation process for most amyloid β-proteins. Repeated freeze-thawing or incubation of amyloid β-protein solutions yielded aggregated samples . To overcome this obstacle, B.A. Yankner et al  utilized a 35 % acetonitrile / 0.1 % TFA solvent system for amyloid β-protein solubilization. This system seemed to be an effective means to achieve initial solubility for most amyloid β-protein fragments, however, aggregation could occur over time in such solutions.
M.P. Mattson et al. reported, that stock solutions of amyloid β-protein (1-38) (H-2966) and amyloid β-protein (25-35) (H-1192) could be prepared by dissolving the peptides at a concentration of 0.5-1.0 mM in water or 1.0 mM DMSO and stored as aliquots at -20 °C until use .
According to S.M. Tomski & R.M. Murphy the solubility of amyloid β-protein (1-40) (H-1194) was a strong function of pH. In PBS (0.14 M NaCl, 0.01 M KH2 PO4/K2 HPO4, 0.02 % (w/v) sodium azide) the peptide was insoluble below pH 7.0. Below pH 3.0 or above pH 7.2 the peptide was soluble, in the latter case up to a concentration of 16 mg/ml. Furthermore, it was asserted that the solubility was not a significant function of ionic strength. As the salt concentration dropped to 75 mM at pH 7.4, the solubility limit remained virtually constant at 15 mg/ml. In deionized Milli-Q water, amyloid β-protein (1-40) was soluble up to nearly 20 mg/ml. In 0.15 M PBS, pH 7.4, at peptide concentrations below 3 mg/ml, no precipitate, turbidity, or gelation was observed, even 3 month after preparation. Above this concentration, although the peptide was originally soluble, a clear continuous gel could be formed, whereby the time for gel formation varied from about 3 weeks at 3 mg/ml to about 2.5 d at 10 mg/ml .
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