Custom peptide synthesis

Process FAQs

• How do we measure the concentration of peptides and proteins?

  Determining the concentration of peptides or proteins can be achieved by either measuring the UV absorbance at 280 nm, reacting the proteins or peptides with dyes and/or metal ions in colorimetric assays (e.g., Bradford assay, Lowry assay, BSA assay) or by performing amino acid analysis, the latter being considered as the gold standard method.

  Amino acid analysis is usually performed when the concentration cannot be determined by UV absorbance due to the lack of aromatic tryptophan and tyrosine residues in the proteins or peptides. Although amino acid analysis requires specialist equipment, it is a robust and highly sensitive method that quantifies the concentration of each individual amino acid in a sample. The protein or peptide concentration is therefore obtained by summing up the concentrations of the single amino acids. Amino acid analysis also has many advantages over colorimetric assays that can suffer from interferences when high concentrations of detergents or chaotropic agents are used. In addition, the dyes/metal ions in colorimetric assays may preferentially react with certain amino acids, whereas the amino acids react equally during the derivatisation step when performing amino acid analysis.

  At AltaBioscience, we provide a comprehensive peptide analysis service. In addition to our peptide synthesis service, we also offer amino acid analysis for the absolute protein quantification of proteins and custom peptides. Our amino acid analysis service is accredited to ISO/IEC 17025:2017.

• How do you generally purify your peptides?

  Peptides are purified using high-performance liquid chromatography (HPLC) to achieve the desired purity level. This technique separates the target peptide from synthesis by-products and other impurities based on differences in their affinity to the chromatography media. 

• What purity is required for custom peptides?

  At AltaBioscience, we provide various purity options ranging from crude-desalted to over 95% purity. Peptide synthesis may generate impurities alongside the desired peptide, such as truncated sequences, deletion sequences, incomplete deprotected sequences, racemised peptides or compounds from side reactions such as aspartimide formation or cysteine oxidation.

  When ordering custom peptides, it is important to check whether the requested peptide purity level will suit the intended research applications. Crude peptides, without any purification, such as those obtained from the Episcan scale, are suitable for screening studies where a large number of peptides are initially tested before undertaking further studies. Standard purification to 80%-95% is suitable for applications such as polyclonal antibody generation and in-vitro assays, and peptide blocking studies, whereas higher purity over 95% is usually required for NMR studies, X-ray crystallography, and enzyme and structural studies.

  Our peptide chemists can guide suitable purity levels for your required application. Contact our experts or learn more peptide purification and peptide analysis.

• What purity do I need for my application?

  At AltaBioscience, we provide various purity options ranging from unpurified to over 95% purity. Peptide synthesis may generate impurities alongside the desired peptide, such as truncated sequences, deletion sequences, incomplete deprotected sequences, racemised peptides or compounds from side reactions such as aspartimide formation or cysteine oxidation.

  When ordering custom peptides, checking whether the requested peptide purity level will suit the intended research applications is important. Crude peptides, without any purification, such as those obtained from the Episcan scale, are suitable for screening studies where a large number of peptides are initially tested before undertaking further studies. Standard purification to 80%-95% is suitable for applications such as polyclonal antibody generation, in-vitro assays, and peptide blocking studies, whereas higher purity over 95% is usually required for NMR studies, X-ray crystallography, and enzyme and structural studies.

  Our peptide chemists can recommend suitable purity levels for your application. Contact our experts or learn more about peptide purification and peptide analysis.

• What does 95% purity mean for my peptide?

  When we say a peptide is 95% pure, it indicates that 95% of the material is the target peptide sequence. The remaining 5% comprises impurities such as truncated sequences, deletion sequences, incompletely deprotected sequences, peptides resulting from side reactions, or other impurities. These are common by-products or residuals from the synthesis process. 

• What is the purity of “crude”?

  AltaBioscience deems peptides with purity levels below 70% as crude. Crude peptide preparations are useful for preliminary studies or applications where high purity is not critical, as they are less costly and faster to produce than purified peptides. However, further purification is usually necessary to isolate the target peptide from the mixture of other components for detailed biochemical studies or therapeutic applications.

• What is net peptide content?

  It is crucial to distinguish between net peptide content and total peptide content. The dry peptide powder you receive typically consists not only of peptides but also other substances such as residual water, absorbed solvents and counterions. The total peptide content (or gross weight) refers to the weight of this entire mixture. In contrast, the net peptide weight represents the actual weight of the peptide itself within the sample. Net peptide content typically ranges from 50-80% of the gross weight. It is different from purity, which specifically measures the percentage of the target peptide sequence within the peptide component of your sample. Our amino acid analysis service can accurately quantify the net peptide content of your peptides. Contact our experts at info@altabioscience.com for more information.

• How do you calculate theoretical net peptide content

  Theoretical net peptide content (calculated assuming that counterions are the only non-peptide components present in your peptide sample) can be estimated by dividing the molecular weight of the peptide by a sum of its molecular weight and the number of trifluoroacetate counterions required to neutralise the peptide multiplied by the molecular weight of the TFA counterion (MW= 114).

  Theoretical net peptide content (%) = MW/ (MW+114x) with x the number of TFA counterions.

  In practice, peptides also contain residual water and absorbed solvents, so the net peptide content is usually determined by quantitative amino acid analysis for accurate results. 

• How do I accurately measure the concentration of peptides and proteins?

  Determining the concentration of peptides or proteins can be achieved by measuring the UV absorbance at 280 nm, reacting the proteins or peptides with dyes and/or metal ions in colorimetric assays (e.g., Bradford assay, Lowry assay, BSA assay), or by performing amino acid analysis, the latter being considered as the gold standard method. 

  Amino acid analysis is usually performed when UV absorbance cannot determine the concentration due to the lack of aromatic tryptophan and tyrosine residues in the proteins or peptides. Although amino acid analysis requires specialist equipment, it is a robust and highly sensitive method that quantifies the concentration of each individual amino acid in a sample. The protein or peptide concentration is therefore obtained by summing up the concentrations of the single amino acids. Amino acid analysis also has many advantages over colorimetric assays that can suffer from interferences when high concentrations of detergents or chaotropic agents are used. In addition, the dyes/metal ions in colorimetric assays may preferentially react with certain amino acids, whereas the amino acids react equally during the derivatisation step when performing amino acid analysis. 

  At AltaBioscience, we provide a comprehensive peptide analysis service. In addition to our peptide synthesis service, we offer for the absolute protein quantification of proteins and custom peptides. Our amino acid analysis service is accredited to ISO/IEC 17025:2017. 

• How stable is a peptide?

  Peptides are generally stable at room temperature for up to a few weeks. For long-term storage, it is best to keep them in a dry and dark place at -20°C. Once reconstituted, peptides should be used within a few days. The stability can decrease if peptides are kept in solution or at room temperature. Protect peptides from light and moisture to maximise stability. 

• What type of chemistry do you use?

  We use solid-phase peptide synthesis (SPPS) to assemble peptide chains, employing Fmoc chemistry.

• What direction are the peptides synthesised?

  Peptides are synthesised from the C-terminus to the N-terminus of the sequence. 

• What is the maximum length of peptide you can produce?

  We have produced peptides containing over 100 residues. However, this is sequence-dependent, and long peptides can be difficult to synthesise. We can assess them on a case-by-case basis and attempt synthesis by fragmentation and ligation. 

• Why are highly hydrophobic peptides difficult to produce?

  Highly hydrophobic peptides present significant challenges in production primarily due to their tendency to aggregate and strong adherence to chromatographic columns. This aggregation complicates the synthesis process as these peptides can form clusters resistant to conventional synthesis techniques. Additionally, their strong affinity for the material in chromatography columns makes them difficult to purify. The adherence complicates the separation process and increases the risk of peptide loss and contamination, further impeding the efficient production of pure peptides. These properties necessitate specialised methods and conditions to synthesise and purify highly hydrophobic peptides effectively. 

• Can you synthesise peptides with several consecutive proline residues

  Proline is a unique proteinogenic amino acid with a cyclic structure. Its nitrogen atom is locked within a five-member ring, and peptides containing at least three consecutive proline residues are known to form polyproline helices, a feature that facilitates protein folding. Synthesis can be challenging due to steric hindrance and the slow rate of peptide bond formation, but our chemists have experience synthesising such peptides. Contact info@altabioscience.com to discuss your requirements.  

• Can you make peptides with N-terminal glutamine?

  Peptides synthesised with an N-terminal glutamine residue are usually unstable during cleavage. Under acidic conditions, they may undergo cyclisation and form pyroglutamate, a five-membered ring lactam. We can acetylate the N-terminus, remove or substitute the N-terminal glutamine, or add another amino acid after glutamine to avoid this unwanted reaction. 

• How can the hydrophilicity of peptides be improved?

  To enhance the hydrophilicity of peptides, you can incorporate polyethylene glycol (PEG) or add three to four lysine (Lys) residues to the sequence. These modifications can significantly increase the peptide’s solubility in aqueous solutions. 

• How do you choose the best antigen for antibody generation?

  Choosing the best antigen for antibody production involved analysing the target protein’s sequence to identify immunogenic regions. These regions should be surface-accessible and hydrophilic. The selected peptides are then synthesised with key modifications, such as conjugated to a carrier protein to enhance the immune response. 

• How can I enhance the cell permeability of my peptide?

  Enhancing the cell permeability of peptides can be achieved through various strategies aimed at improving their ability to cross the cell membrane. At AltaBioscience, we can incorporate sequences known as cell-penetrating peptides, such as Tat peptide or penetratin, into your peptide sequence. These peptides have inherent cell-penetrating properties and facilitate the internalisation of cargo molecules into cells. Another strategy is to conjugate lipid moieties (e.g., fatty acids) to the peptide sequence, which can enhance membrane interactions and promote cell uptake.

• How can I increase peptide stability in cells?

  To increase peptide stability in cells, you can introduce chemical modifications such as D-amino acids, acetylated N-terminus and amidated C-terminus to enhance resistance to proteases. Forming cyclic peptides can also improve stability by reducing conformational accessibility. Additionally, incorporating non-natural amino acids can further protect the peptide from degradation.