FAQs

To request a quote for catalog peptide, or custom peptide, please submit your information by clicking here or by: email (info@biopeptek.com), phone (610-643-4881), toll free (800-352-6169) or fax (610-910-3487). Please provide the appropriate information as follows:

• Catalog peptide: Catalog number and quantity (mg)
• Custom peptide: Amino acid sequence and modifications, quantity (mg), and purity (Desalted, >75%, >80%, >90%, >95%, >98%)

All peptides are analyzed by MS to confirm the molecular weight. For peptides where a minimum purity has been requested we also run reverse-phase HPLC analyses. The results from these analyses are included on the Certificate of Analysis (COA) supplied with the peptides delivered. In addition, product color and appearance and amount (mg) information is also included in the COA.

The typical turn-around time is 2-3 weeks for a standard peptide under 30 amino acids. This time could vary depending on the length and difficulty of the peptide. Upon initiation of our project, we will discuss timing of the project so you are aware of the projected delivery date.

Typical peptide purities are as follows.

1. Peptide purity >85%: immunological applications, polyclonal antibody production and non-sensitive screening.
2. Peptide purity >90%: Structure-Activity Relationship and Bioassays.
3. Peptide purity >95%: ELISA, enzymology, and biological related activities.
4. Peptide purity >98%: Structural studies/Crystallography, NMR and sensitive bioassays.

Peptide purity is determined by high performance liquid chromatographic (HPLC) method based on the peptide relative percent area using UV absorbance at a wavelength of 214nm. The synthetic peptide purity value obtained by this method does not include water and trifluoroacetate salt which are present in the lyophilized material since they do not have UV absorbance at 214 nm.

Unless specified in the order, all lyophilized peptides supplied by Biopeptek contain a variable amount of water and a small amount of trifluoroacetate as the synthetic peptide counter-ion. Acetate or chloride can be supplied upon request.

Net peptide content is determined by quantitative amino acid analysis, which measures the amount of each amino acid using acid hydrolysis method. The synthetic peptide content is then calculated based on the total number of amino acids present in the peptide. Generally, net synthetic peptide content is in a range of 70% to 90%, and in some cases, it can be as low as 30%.

Biopeptek provides different levels of custom peptide purity ranging from crude to 98% for different applications. A general guideline for choosing custom peptide purity is described below.

Crude peptide to <75%: Non-sensitive screening assays. (Desalted to 75% is recommended).

Peptide >75%: Immunological applications, polyclonal antibody production ELISA standard for measuring titers of antibodies Affinity chromatography Peptide array production

Peptide >85%: Immunological applications Non-quantitative enzyme-substrate studies Phosphorylation assays Non-quantitative peptide blocking studies Coupling to resins for affinity purification Coating of tissue culture plates for cell attachment Protein electrophoresis applications

Peptide >95%: Quantitative Receptor-ligand interaction In vitro bioassays In vivo studies Quantitative blocking and competitive inhibition assays Quantitative phosphorylation studies NMR studies Physical properties standards Structural studies Enzyme studies Monoclonal antibody production

Peptide >98%: SAR Studies Clinical trials API (Active Pharmaceutical Ingredients) Commercial products X-Ray crystallography studies Commercial products

At Biopeptek, custom peptides are synthesized using solid phase peptide synthesis (SPPS) and liquid (solution) phase peptide synthesis methods followed by purification processes. The entire process is in compliance with current Good Manufacturing Practice (cGMP) to ensure the custom peptide quality. The diagram below depicts our quality system at Biopeptek.

A synthetic peptide contains two major non-peptide impurities: dithiothreitol (DTT) and trifuoroacetic acid (TFA). DTT is toxic to cells. If custom peptides are used for cell culture studies, the tolerate DTT level is less than 1M for most cells. For crude peptides that go through the common ether precipitation process, the residual amount of DTT should be relatively small. For purified synthetic peptides that have gone through a purification process, the DTT level is negligible.
Most custom peptides are purified using the solvent with a small amount of TFA, which can be removed after lyophilization for at least 48 hours, but TFA salts can be difficult to remove using lyophilization step alone. If TFA or TFA salts are suspected to cause problems in the experiment, it should be notified in the order, and special steps will be taken to carry out the salt exchange.

• The peptide-containing vial should be tightly capped at all times. The experiment should be carefully planned to minimize opening of the lyophilized peptide vial.
• Short term storage (<3 months) of lyophilized peptide should be done in a freezer at -202103. For longer storage, we recommend storing the peptide in a deep freezer at -802103 if available.
• Repeated freeze-thaw cycles should be avoided for both lyophilized peptides and peptide solutions. If custom peptide samples need to be frequently or periodically taken from the stock, it is recommended to make a series of aliquots from the stock.
• Synthetic peptide sequences containing C, M, or W 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.
• In general, peptide solutions are stable for up to a week at 42103. However, if the synthetic peptide sequence has inherent instability (for example: containing C,M,W or D-G-,-D-P- structure), it might be better to freeze the solution when not in use. Peptide solutions at pH>8 should also be frozen when not in use.

Solubility is often a major challenge for researchers who work with synthetic peptides. Because each amino acid exhibits unique physical characteristics, each synthetic synthetic peptide has specific solubility requirements. Dissolve synthetic peptides in an appropriate buffer: acidic peptides in basic buffer and basic peptides in acidic buffer. If necessary, sonicate briefly.

• Custom Peptides containing Trp, Met or Cys require special care to avoid oxidation. Oxygen-free water or reducing agents may be used.
• In general, synthetic peptide solutions are stable for up to a week at 42103. However, if the synthetic peptide sequence has inherent instability (for example: containing C, M, W, N, Q or D-G-, -D-P- structure), it might be better to freeze the solution when not in use. Custom peptide solutions at pH>8 should also be frozen when not in use.
• Most custom peptides containing Trp, Met, Cys, Asn or Gln have limited shelf life. Long-term storage is not recommended.

The steps outlined below provide you with a method for determining the best solvent for a synthetic peptide based on its amino acid sequence. It is best to first solubilize a small aliquot of the sample, rather than the entire stock.

• Assign a value of -1 to each acidic residue. The acidic residues are Asp (D), Glu (E), and the C-terminal -COOH. Assign a value of +1 to each basic residue. The basic residues are Arg (R), Lys (K), His (H), and the N-terminal -NH2.
• Calculate the overall charge of the synthetic peptide.
• If the overall charge of the synthetic peptide is a positive value, you have a basic custom peptides. Initially try to dissolve the peptide in water. If the peptide does not dissolve, try 10% and higher solutions of acetic acid. If the peptide still does not dissolve, add TFA (<50?L) to solubilize the peptide and dilute to 1mL with deionized water.
• If the overall charge of the peptide is a negative value, you have an acidic peptide. Initially try to dissolve the peptide in water. If the custom peptides does not dissolve, add NH4OH (<50?L) and dilute to 1ml with deionized water.
• If the overall charge of the synthetic peptide is zero, your custom peptides is considered neutral. Neutral peptides may require the addition of organic solvents, such as acetonitrile, methanol, or isopropanol. The addition of denaturants, such as urea or guanidinium-HCL may also be necessary.

If a synthetic peptide has a purity of 90%, the other 10% contains synthetic peptides that have shorter sequences, truncated sequences, sequences with incomplete deprotectionly deprotected sequences

• Peptide sequences modified during cleavage (reattachment of protecting groups at other locations on the synthetic peptide)
• Synthetic peptides that have undergone side reactions

The steps outlined below provide you with a method for a good idea to choose the terminal ends of the custom peptide dependent on the natural occurrence of the peptide sequence:

• The custom peptide should mimic an internal sequence of a protein. The peptide should not be charged at the ends. The N-terminus of the custom peptide should be acetylated and the C-terminus should be amidated.
• If the synthetic peptide sequence is the C-terminal end of a protein, the C-terminus should be the free acid and the N-terminus should be acetylated.
• If the synthetic peptide sequence is the N-terminal end of a protein, the C-terminus should be an amidated and the N-terminus should be in the natural free amine form.
• If the custom peptide is for cytotoxic T-cell epitope studies, a free amino group at the N-terminus and a free acid at the C-terminus are necessary. These ends are the natural equivalents to the synthetic peptide fragments, processed intracellularly from whole proteins.
• If the custom peptide is for antibody production, please check the technical resources section of our website.

Both KLH and BSA are common carrier proteins which are long peptides. The molecular weight of BSA is much smaller than KLH. However, BSA is much more soluble and immunogenic. It contains 59 lysines, 30-35 as primary amines capable of reacting with conjugation sites of linkers. It is a popular carrier for weakly antigenic compounds. BSA can be used to block nonspecific binding sites in many immunochemical experiments such as ELISA, immunoblotting and immunohistochemical studies. It may be used as a non-relevant protein in enzyme immunoassays.

The KLH cannot be used for this because the anti-KLH antibodies, which formed during immunization, will interfere with the measurement of anti-heptan antibodies. When KLH is used as the carrier, heptan-BSA conjugates can be used because they do not interfere with the measurement for anti-heptan antibodies.

Biopeptek routinely peptide synthesis of 3 – 50 amino acids in length. We can also synthesize peptides of greater than 100 amino acids in length. The minimum length of synthetic peptides should not be less than 3 amino acids. For shorter custom peptide sequences, cleavage from the synthesis resin and following purification can be problematic.

Solid phase peptide synthesis is carried out under a controlled and calculated environment. There are very few occasions that the synthetic peptide sequence is in question. By using a mass spec analysis, you can determine the molecular weight of the synthetic peptide, thus proving the sequence completion of the peptide synthesis. However, there are cases where peptide sequencing is something we recommend, such as with MAPS peptides. With the majority of MAPS peptides, the mass spec analysis provides inconclusive information, because of the nature of MAPS peptides. Sequencing can be an easy alternative for proof of peptide synthesis completion, and affirmation of peptide sequence in publication use.

Polyethylene glycol (PEG) is a useful delivery system for custom peptide and protein-based biopharmaceuticals. PEGylation is the chemical attachment of PEG to a custom peptide on a specified site of the molecule. Studies have shown an increase in the potential bioavailability of custom peptides when incorporating PEG into synthetic peptide sequences versus the injection of a naked peptide. Drug oriented custom peptides show significant improvement to their therapeutic properties, including better patient compliance and side effect profile.