Peptide Synthesis FAQs

For quotation, who should I contact?

You can email or contact us as below:

Singapore peptide@axilscientific.com +65 6775 7318
Malaysia and other countries  info@apicalscientific.com or contact our respective sales representative in your region. info@apicalscientific.com or contact our respective sales representative in your region.

 

For quotation, what information do you need? 

We need to have your peptide sequences in order to issue quotation. If there is any complexity involved in synthesizing the peptide, difficulty charges will be imposed.
Customers are recommended to fill up our peptide order form and email to our respective customer care department to request quotation directly.
If peptide sequences are not available, we need the minimum information, i.e. number of amino acid (a.a.), purity (in HPLC %), amount of peptide (in mg) and modification (if there is any) to estimate the quotation.

 

Do you synthesize short sequence peptides? 

Yes, we can synthesize short peptides as small as 2 amino acids or named as dipeptides.

 

Do you synthesize peptides with modification? 

We offer a couple of modification to peptide synthesis. They are phosphorylation, biotinylation, cyclization, fluorescein, D Amino Acid and conjugation.

 

What are the deliverables (eg. reports, etc.)?

We will provide: 
  1. Peptide Synthesis Summary
  2. HPLC or Mass Spectrometry QC Report
 
What is the Turn-Around-Time (TAT)? 
 
The synthesis may take approximately 3 – 4 weeks. If there is any complexity of the peptide sequence, it may take additional 1 to 2 weeks.
 
 
Can you aliquot the peptides into smaller quantities? 
 
Yes, we do aliquot peptides into small quantities for a small fee. Such request must be confirmed before the synthesis begins.
 
 
What is the maximum peptide length that your company can produce?
 
We can synthesize up to 120 amino acids in length and in various purity (crude, 70% to 99% of purity). Full peptide sequence is required to check for complexity.
 
 
What should I consider while designing my custom peptide?
 
a. Solubility 
 
It is important to consider if the peptide will be soluble enough for your application and/or soluble in HPLC compatible solvents to allow for its purification to the desired degree of purity. Generally, peptides composed of predominantly hydrophobic amino acids (W, Y, F, L, I, V, M, C, A) tend to be less soluble in aqueous solvents. Hydrophilic amino acids (R, K, H, S, T, D, or E) help in peptide solubility, dependent on their prevalence in the sequence.
 
b. Potential synthesize difficulties 
 
While most peptides can be synthesized and purified successfully, sometimes minor modifications to the sequence may greatly improve the chance of a successful synthesize and could shorten the manufacturing or purification process or even lower its cost. To minimize synthesize difficulties, please email us your sequences so that we can run a quick analysis to identify likely synthesize difficulties and suggest alternatives.
 
c. Labeling of peptides 
 
Attachment of various labels (biotin, fluorescein, etc.) to the peptide is the most straightforward when these labels are to be attached at the N-terminus. Attachment at the C-terminus or in the middle of the sequence is more demanding and more expensive.
 
What is the required purity of the peptides for my application?
 
Crude peptides may contain a large amount of impurities such as residual solvent, trifluoroacetate (TFA) and other truncated peptides. Hence it is not recommended for use in biological assay. Typically, peptides are delivered as TFA salts. However, in cases, TFA residual may present issue in the subsequent application. For such case, alternative salt such as acetate and hydrochloride can be considered. These salt form are usually 20-30% more costly compared to TFA salts due to the greater peptide loss in the salt conversion process, but it can help to eliminate the concerns on the presence of TFA in the downstream process particularly for biological assay. 

We recommend the following levels of peptide purity for various applications as below.

Percentage of Purity Application
70-98% Immunograde purity, antibody production
80-98% Non-sensitive screening assays
90-98% Enzymology, bioactivity studies
95-98% Bioassays receptor-ligand studies, structural studies

 

What is the IUPAC amino acid code?

Alanine Ala A Methionine Met M
Cystein Cys C Asparagine  Asn N
Aspartic Acid Asp D Proline  Pro P
Glutamic Acid Glu E Glutamine  Gln Q
Phenylalanine Phe F Arginine  Arg R
Glycine  Gly G Serine  Ser S
Histidine  His H Threonine  Thr T
Isoleucine  Ile I Valine  Val V
Lysine  Lys K Tryptophan  Trp W
Leucine  Leu L Tyrosine  Tyr Y

 

How do I know if my custom peptide will be soluble?

The solubility of a peptide in water cannot be predicted based on its structure. There are some basic characteristics to predict solubility.
 
a. Peptide with <5 amino acids can be easily soluble in aqueous solution. However, if the entire sequence consists of hydrophobic residues ,it will have only limited solubility or could be completely insoluble.
 
b. Hydrophobic peptides with >25% charges amino acid (such as E, D, K, R and H) and <25% hydrophobic residues are usually soluble in aqueous solution.
 
c. Hydrophobic peptides whose sequence contains ≥50% hydrophobic residues might be completely or only partially soluble in aqueous solutions. These peptides should instead be dissolved in organic solvents such as DMSO if they do not contain C, W, or M residues. If they do contain these amino acids, they should be dissolved in DMF, acetonitrile, isopropyl alcohol, ethanol, acetic acid, 4–8 M guanidine hydrochloride (GdnHCl), or urea prior to being diluted carefully in aqueous solution.
 
d. Hydrophobic peptides contain a very high proportion (>75%) of hydrophobic amino acid (such as D, E, H, K, N, Q, R, S, T, or Y) can form intermolecular hydrogen bonds (cross-links), which eventually causing gel formation in concentrated aqueous solutions. Therefore, peptides should be dissolved in an organic solvent or strong solvent like TFA or formic acid that is compatible with the final experiment. After dissolved, the solution can slowly be added by drop-wise to a stirring aqueous buffered solution. Peptide might precipitate when added to an aqueous buffered solution. Thus, the limit of solubility is reached when the resulting peptide solution begin to show turbidity.
 
 
How to store and handle my custom peptides?
 
All peptides are delivered lyophilized.
 
a. Opening the package:
 
It is better to equilibrate the peptide at room temperature in a desiccator prior opening and weighing. Please take note that failure to warm the peptide beforehand will cause the formation of condensation (peptides tend to be hygroscopic) on the product when the bottle is opened which eventually result in reducing the stability of the peptide. Before reconstitution, centrifuge the vial of lyophilized peptide at 12,000 x g for 20 seconds. This will help pellet the entire peptide sample for reconstitution. 
 
b. Weighing peptides:
 
Weigh out your required quantity of peptide rapidly and store the rest of unused peptide at -200oC or below. Please take note that the peptides sequence contains C, M, W, N, Q and E will have a shorter shelf life than other peptides. 
 
c. Storage guidelines for lyophilized peptides:
 
  1. The peptides should remain sealed and be kept under cool, dry condition & away from light.  For short term storage such as a few days to 2 months, the lyophilized peptides should be stored at 40oC.
  2. For long term storage, we recommend to store the peptide in a deep freezer at -800oC. This will keep the peptide stable for years; however the contamination with moisture will greatly affect the long term stability of peptide. If the peptides need to be frequently taken from the stock, it strongly recommended to make a series of aliquots from the stock.
  3. We suggest customer to aliquot into few tubes and store at freezer to avoid contamination and frequent repetitive freeze-thaw cycle of the peptides.     
 
d. Storage guidelines for peptides in solution:
 
The shelf life for peptide in solution form is limited, especially for the peptides which contain Cysteine (C), Methionine (M), Tryptophan (W), Asparagine (N), Glutamine (Q) and N-Glutamic Acid (E) are prone to air oxidation. It is recommended to purge the air out of the vial and replace it with a blanket of nitrogen or argon. If storage in solution form is unavoidable, use sterile buffers around pH 5 to 6 and freeze aliquots for storage at -200oC to prolong the storage life of peptide in solution. Furthermore, avoid the use of frost-free freezer as there will be variations in temperature during the frequent defrosting cycle. Repeated freeze-thaw cycles can also damage the peptides.
 
 
How should I dissolve the peptides? 

The solubility of a peptide is dependent mainly on its polarity. The following are common solvent use to dissolve the peptide. 

Type of Synthetic Peptide Solvent
1. Acidic peptides Basic buffer
2. Basic peptides Acidic solutions
3. Hydrophobic peptides & neutral peptides (contain large number of hydrophobic or polar uncharged amino acids) DMSO, DMF, acetic acid, acetonitrile, methanol, propanol, or isopropanol, and then diluted using water.

[Note 1: For (3), dissolve in small amount of the suggested organic solvent and dilute using water.]
[Note 2: DMSO should not be used with peptides that methionine or free cysteine because it might oxidize the side-chain.]

Thus, it is best to first solubilize a small aliquot of the peptide, rather than the entire peptide. It is strongly recommended to test several different solvents until the appropriate one is found. If the peptides persist as visible particles, sonication can be used to break down the lumps of solid peptide to small particles and vigorously stirs the solution to enhance solubility.
a. First, assign value of -1 to each acidic residue. Next, assign a value of +1 to each basic residue and then calculate the overall charge of the peptide. (refer to table below)
 
Charge of Peptide Amino Acid Residue 
Positive (+) K, R, H, and the N-terminus
Negative (-) D, E, and the C-terminus
Hydrophobic uncharged residues F, I, L, M, V, W, and Y
 
b. If the overall charge of the peptide is a positive value, the peptide is basic. Initially, you may try to dissolve the peptide in water. If the peptide still refused to dissolve, try to dissolve the peptide with 10-25% of acetic acid. If the above suggested methods still fail, then add TFA (10 to 50uL) to solubilize the peptide and dilute with distilled water to your desired concentration.
 
c. If the overall charge of the peptide is a negative value, the peptide is acidic. For acidic peptide, it might be soluble in PBS (pH7.4). If the peptide does not dissolve, add a small amount of basic solvent like 0.1M ammonium bicarbonate to solubilize the peptide and then add distilled water to your desired concentration.
[Note: Peptide that contain free cysteines should be reconstituted in de-gassed acidic buffers because thiol moieties will be oxidized rapidly to disulfides at pH>7.]
 
d. If the overall charge of the peptide is zero, the peptide is neutral. For neutral peptide, it might be soluble in organic solvent such as acetonitrile, methanol or isopropanol. 
 
[Note 1: For very hydrophobic peptides, try to add a small amount of DMSO and then dilute the solution with distilled water to your desired concentration.]
[Note 2: For Cys-containing peptides, use DMF instead of DMSO, whereas for the peptides that tend to aggregate, add 6M guanidine, HCI or 8M urea and then dilute to the desired concentration.]
 
 
What are the quality control assurances on our synthetic peptides?
 
All peptides synthesized are provided with QC report, which includes HPLC or ESI Mass Spectrometry chromatograms to ensure purity and mass spectral analysis to confirm identity.