FAQ
Peptide solubility is mainly determined by polarity. Use acidic solutions to dissolve basic peptides and use basic solutions to dissolve acidic peptides. Hydrophobic and neutral peptides containing a large number of hydrophobic or polar uncharged amino acids can be dissolved in organic solvents such as DMSO, DMF, acetic acid, acetonitrile, methanol, propanol or isopropanol. The solutions should be diluted with water. If methionine and free serine are present in the peptide, DMSO should not be used as a solvent in order to avoid oxidation of the side chain.
Best practice is to test a small portion for solubility before dissolving the entire peptide. Until the best solvent is determined, several tests may be necessary. Lyophillized peptides should be briefly centrifuged beforehand to form a pellet. To enhance solubility sonication can be used.
An easy way to determine which solvent should be used for dissolving your peptide is:
- First, the total charge of the peptide should be calculated. For this purpose, the acidic residues (Asp [D], Glu [E], and the C-terminal –COOH) are assigned a value of -1 and the basic residues (Arg [R], Lys [K], His [H], and the N-terminal -NH2) a value of +1. With these values the total charge can be calculated.
- Overall charge is positive: the peptide is basic. Our recommendation is to try to dissolve the peptide in water. If dissolving with water does not work try to add acetic acid (total amount 10-25%). If this does not work either, a small amount of TFA (10–50 µl) should be added and can be further diluted with water to your desired concentration.
- Over all charge is negative: the peptide is acidic. We recommend usage of PBS-buffer (pH:7,4) to dissolve the peptides. If this fails a small amount of basic solvents such as 0.1 M ammonium bicarbonate should be added and can be further diluted with water. For peptides containing free cysteine degassed acidic buffers should be used due to the possibility of oxidation of the thiol group.
- Overall charge is 0: peptide is neutral. Neutral peptides usually dissolve in organic solvents such as acetonitrile, methanol and isopropanol. A small amount of one of these solvents should be added to dissolve the peptide. If the peptide is too hydrophobic we recommend to use a small amount of DMSO and can further be diluted with water. For peptides containing free cysteine DMF instead of DMSO should be used.
- If none of the suggested solvents work, we suggest using trifluoroethanol (TFE). TFE may form a solvent matrix for assisted hydrophobic interactions between peptide side chains and also TFA can induce and stabilize α-helices and can induce β-turns, β-hairpins and also β-strands. However TFA can disrupt tertiary interactions in proteins by weakening non-polar interactions while preserving secondary structures. It has been found that a mixture of TFE or hexafluoroisopropanol (HFIP) with trichloromethane (TCM) or dichloromethane (DCM) also works very well to dissolve peptides. It has been shown that at a content of 10% HFIP or about 20% TFE, clathrate structure is formed.
Positively charged residues: K, R, H, and the N-terminus
Negatively charged residues: D, E, and the C-terminus
Hydrophobic uncharged residues: F, I, L, M, V, W, and Y
Uncharged residues: G, A, S, T, C, N, Q, P, acetyl, and amide
Examples:
His- |
Arg- |
Phe- |
Ala- |
Lys- |
Ser- |
Arg- |
Asp- |
Glu- |
NH2 |
|||
+1 |
+1 |
0 |
0 |
+1 |
0 |
+1 |
-1 |
-1 |
+1 |
(+5)+(-2)=+3 |
this is a basic peptide, see step 2 above |
Asp- |
Arg- |
Gln- |
Tyr- |
Leu- |
Gly- |
Arg- |
Glu- |
Glu- |
OH |
|||
-1 |
+1 |
0 |
0 |
0 |
0 |
+1 |
-1 |
-1 |
-1 |
(+2)+(-4)=-2 |
this is an acidic peptide, see step 3 above |
Pro- |
Lys- |
Thr- |
Val- |
Leu- |
Leu- |
Met- |
Cys- |
Ile- |
OH |
||
0 |
+1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
-1 |
(+2)+(-2)= 0 |
this is an neutral peptide, see step 4 above |
Hydrophobic peptides can be dissolved in DMSO. Peptides dissolved only in DMSO can be cytotoxic and therefore should not be used in cell cultures. For this reason, the amount of DMSO should be kept as low as possible. Slight amounts, from 5% of DMSO can cause cell walls to dissolve. Some cell cultures can tolerate 0.5% and some up to 1% DMSO. A final concentration of 0.5% is often used for cell cultures without showing cytotoxicity. However, less than 0.1% DMSO should be used for primary cells.
We recommend dissolving the peptide in the smallest possible volume of a 50% (v/v) DMSO/water mixture and subsequently add water/buffer until the desired concentration is achieved. If the product precipitates again during this process and cannot be re-dissolved by adding DMSO, then lyophilization is required and re-soluabilize another attempt of dissolving is needed.
Basic buffers should be avoided for peptides containing disulfide bridges. The disulfide bond is formed quickly at a neutral and slightly basic pH. However, the disulfide bond is reversible and can be reduced with DTT at basic conditions. The optimum pH-range is between 7.0-9.5. Due to the easy oxidation of DTT, the solution should be freshly prepared. Peptides containing free thiol groups may oxidize to form dimers or oligomers during storage, including the lyophilized form at a low temperatures. Peptides provided as acetate salts are more sensitive to cysteine oxidation than the corresponding TFA salt or HCl salt.
For long-term storage, we recommend to store the peptides in lyophilized form at -20°C, or preferably at -80°C in sealed containers to minimize peptide degradation. Under these conditions, peptides can be stored for up to several years and this prevents different kinds of degradation such as oxidation, formation of secondary structures and bacterial contamination. The shelf stability of peptides is sequence-dependent. Sequences containing cysteine, methionine, tryptophan, asparagine, glutamine and N-terminal glutamic acid will have a shorter shelf life than other peptides. For short-time storage, we recommend to store the peptides in a refrigerator (+4°C). The peptides should be protected from direct sunlight and peptides with fluorophores should be stored in darkness.
Before opening the peptides, it is better to equilibrate to room temperature as peptides tend to be hygroscopic and can lead to cause condensation. Condensation can reduce the stability of the peptides. Before using the peptides, we recommend centrifuging them.
Dissolved peptides are less stable than lyophilized peptides and shouldn’t be stored long-term. We recommend to lyophilize the dissolved peptides.
Unfortunately, it is not possible to predict if a peptide is water-soluble by studying its structure alone, but there are clues for determining solubility, especially for short peptides. The ε-amino group of Lysine and the guanidine group of Arginine are in pronated form in peptides sold as TFA salts and are easily dissolved in water. Peptides containing many acidic amino acids such as aspartic acid and glutamic acid, however, are difficult to dissolve in water but are readily soluble with ammonia or basic buffers.
Here you will find certain basic characteristics to predict solubility:
- Short peptides with <5 amino acids are usually highly soluble in aqueous solutions. However, if the peptides contain only hydrophobic amino acids, they may be difficult or impossible to dissolve in water.
- Peptides with many hydrophilic amino acids (>25%) (E,D,K,R,H) and few hydrophobic amino acids (<25%) are usually well soluble in aqueous solutions.
- Peptides containing ≥50% hydrophobic amino acids should be well or partially soluble in aqueous solutions. If the peptides are only partially soluble, organic solvents such as DMSO, DMF, acetonitrile, isopropyl alcohol, ethanol, acetic acid, 4-8 M guanidine hydrochloride (GdnHCl), or urea should be used and further diluted with aqueous solutions. However, peptides containing C, M or W should not be dissolved in DMSO due to oxidation reactions.
- Hydrophobic peptides with more than 75% hydrophobic amino acids are usually not soluble in water. For these peptides, strong solvents such as TFA or formic acid should be used for dissolution. Dilution with an aqueous solution could again lead to precipitation of the peptide. Therefore, high concentrations of organic solvents or denaturants may be necessary to completely dissolve the peptide.
- Peptides containing a very high proportion (>75%) of the following amino acids: D, E, H, K, N, Q, R, S, T, or Y can form intermolecular hydrogen bonds, which leads to gel formation in aqueous solutions. Such peptides are best dissolved with organic solvents. Dilution with aqueous solutions should only be done dropwise. The limit of aqueous solubility can be recognized by the turbidity of the solution.
p&e offers different purity levels starting with crude up to >95%. Ultra-pure materials with >97% purity are available upon request.
Crude peptides are not recommended for biological assays. Crude peptides may contain non-peptide impurities such as residual solvents, scavengers from cleavage, TFA and truncated peptides. TFA cannot be totally removed for crude peptides.
Peptides are usually delivered as TFA salt.
The following list might serve as guideline for peptide specification:
crude - 70%: Peptide arrays, Antigens for antibody production, Competitive elution chromatography, ELISA standards for measuring antisera titers, immunological applications, protein-protein interactions, sequence optimization, epitope discovery and no sensitive screening;
>80%: immunological applications and epitope validation; Western blotting studies (non-quantitative), Enzyme-substrate studies (non-quantitative), Peptide blocking studies (non-quantitative), Affinity purification, Phosphorylation assays, Protein electrophoresis applications and immunocytochemistry
>90%: immune monitoring assays
>95%: NMR studies, crystallization, enzymatic assays, quantitative studies of receptor-ligand interactions
>97%: NMR studies, crystallization, sensitive bioassays, API (Active Pharmaceutical Ingredients)
The gross weight includes the weight of the peptide plus the counter-ion and the attached water.
The net peptide content is different from the peptide purity. The net peptide content is the percentage of peptide relative to non peptidic materials, mostly counter ions and moisture. The net peptide content can be determined by amino acid analysis. Please place a request for a quote if you require this service. This analyses will be conducted by an independent DIN EN ISO/IEC 17025 accredited test laboratory.
Usually, hydrophilic peptides absorb tiny amounts of moisture even under strict lyophilized conditions. Net peptide content may vary from batch to batch depending on the purification and lyophilization processes.
Theoretical net peptide content (calculated assuming that counter ions are the only non-peptide components present in your peptide sample) can be estimated by dividing molecular weight of the peptide (MW peptide) by a sum of this molecular weight and a number of the counter ions (N) that are required to neutralize the peptide multiplied by the molecular weight of the counter ion (MW (TFA)= 114 Da, MW (HCL)= 35,5 Da, MW (acetate)=59 Da).
For example, a synthetic peptide of MW= 1000 with a free N-terminal amino group and one Lys has theoretical net peptide content of 1000 / (1000 + (2 x 114)) = 1000/1228 =0.81 or 81%. Counter ions are not the only potential non-peptide components in the peptide sample. They can also contain residual water, adsorbed solvents and traces of other substances. As a result, the actual net peptide content is usually determined by either elemental analysis (N2 content) or quantitative amino acid analysis. It should be noted that arginine can bind more than 1 eq. of TFA.
Synthetic peptides are manufactured by solid-phase synthesis. TFA is usually used for cleavage and purification steps. TFA binds to the free amino termini and side chains of positively charged amino acids. The TFA counter ion could change the secondary structure, mass, solubility of the peptide, or results of in vivo studies. TFA is toxic in cell experiments. Hydrochloride is suggested for hydrophobic peptides and acetate is generally considered as pharmaceutically acceptable counter ion.
All peptides from p&e are lyophilized to easily remove excess and unbound TFA. However, HPLC and salt exchange is required to remove the TFA counter ions that are binding to the positively charged peptide residues. The most adapted method is to replace TFA counter ions by a stronger acid such as hydrochloric acid (HCl).
To exchange TFA for acetate, the ion exchange method is used. First, the TFA anion is exchanged for the chloride using an Amberlite IRA402 resin. This is followed by a further ion exchange first into OH and then directly into acetate.
Upon request, p&e can aliquot part or all of your order into smaller quantities for a minimum fee of 2,50 € per tube. Aliquoted products are more expensive but may save you time, effort and money during the determination of peptide solubility. Your peptides will also be more stable because they will not be exposed to multiple freeze-thaw cycles, openings and closings of the container, mishandling, or bacterial contamination. Peptide oxidation, degradation, and aggregation are less prevalent in aliquoted samples.
Chemically synthesized peptides carry free amino and carboxy termini. The need for N-terminal acetylation or C-terminal amidation must be stated explicitly during ordering. It is impossible to perform these modifications after synthesis has been completed.
N-terminal acetylation and C-terminal amidation reduce the overall charge of a peptide and decrease solubility. However, the stability of the peptide usually increases because the terminal acetylation and amidation allow the peptide to mimic the native protein more closely and prevent enzyme degradation. In this way, these modifications may increase the peptides' biological activity.
5-carboxy-fluorescein (5-FAM, 5-Fluo) |
ex. 493 nm/ em.517 nm |
Emission color: green (dark) |
6-carboxy-fluorescein (6-FAM, 6-Fluo) |
ex. 495 nm/ em.517 nm |
Emission color: green (dark) |
5,6-carboxy-fluorescein (5,6-FAM) |
ex. 495 nm/ em.517 nm |
Emission color: green (dark) |
Fluorescein Isothiocyanate |
ex. 490 nm/ em.520nm |
Emission color: green |
Tetramethyl rhodamine (TAMRA) |
ex. 544 nm/ em.576 nm |
Emission color: green-yellowish |
Cyanine 3 (Cy3) |
ex. 550 nm/ em.570 nm |
Emission color: green |
Rhodamine B |
ex. 546 nm/ em.568 nm |
Emission color: green |
Cyanine 5 (Cy5) |
ex. 650 nm/ em.670 nm |
Emission color: red |
We provide other dyes for dye-labelled peptides on request.
Most of p&e’s products will be shipped at room temperature in sealed bags or boxes. The peptides are provided in 2mL vials. Upon request, p&e offers shipment on dry or blue ice with or without temperature tracking. For special requests please contact us.
Peptide pools consist of individual 15mer peptides, each overlapping by 11 amino acids. These synthesised and pooled peptides comprise complete protein antigens.
for T cell epitope identification?
Just send us the UniProt ID or the sequence of the antigen to get a quote for your customized peptide pool. Contact: lips@peptides.de
DMSO is the best solvent to dissolve peptide pools however a concentration of more than 1% DMSO should not be exceeded. A higher concentration of DMSO could lead to cell death of cell cultures. After dissolving the peptides with DMSO, dilution with a suitable buffer system should follow.
Yes, here you can download the detailed protocol for regeneration of the membrane: more Info >
We offer for Peptide Arrays the following membranes:
Beta-Ala, CAPE and TOTD
Acetylation is relevant for peptides of different lengths in a Spot-Synthesis, otherwise, there is a risk of contamination by activated amino acids, which are coupled to the longer peptide chains.
Yes, spot synthesis allows unnatural and modified amino acids, including the D and L forms.
Yes, we offer both biotinylated and disulfide cyclized peptides.
The offset should not be longer than 3 amino acids, otherwise the grid is too coarse. The finer the grid, the higher the accuracy of information can be obtained.