Peptides shipped at room temperature are highly stable at lyophilized form in sealed bags.Peptides should not be kept in solution for long periods.

Peptide storage guidelines:
For long-term storage, peptides should be stored in lyophilized form at -20°C or preferably at -80°C with desiccant in sealed containers in order to minimize peptide degradation. Under these conditions, peptides can be stored for up to several years. This type of storage prevents bacterial degradation, oxidation, and the formation of secondary structures.

Opening the package:
It is better to equilibrate the peptides to room temperature in a desiccator prior to opening and weighing. Failure to warm the peptides beforehand can cause condensation to form (peptides tend to be hygroscopic) on the product when the bottle is opened. This will reduce the stability of the peptide products.

Weighing peptides:
Weigh out your needed quantity of peptide quickly and store all unused peptide at -20°C or below. Sequences containing cysteine, methionine, tryptophan, asparagine, glutamine, and N-terminal glutamic acid will have a shorter shelf lives than other peptides.

The solubility of a peptide is determined mainly by its polarity. Acidic peptides can be reconstituted in basic buffers and basic peptides in acids. Hydrophobic peptides and neutral peptides containing large numbers of hydrophobic or polar uncharged amino acids should be dissolved in a small amounts of organic solvent such as DMSO, DMF, acetic acid, acetonitril, methanol, propanol or isopropanol and then diluted with water. DMSO should not be used with peptides containing methionine or free cysteine because it may oxidize the side-chain.

Test a portion of the synthesized peptide before dissolving the rest of the sample. You may need to test several different solvents until you find an appropriate one. Sonication enhances solubilization.

• 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. Then calculate the overall charge of the peptide.


• If the overall charge of the peptide is positive, the peptide is basic. Try to dissolve the peptide in distilled water if possible. If it fails to dissolve in water, then try to dissolve the peptide in a small amount of 10-25% acetic acid. If this fails, add TFA (10-50 μl) to solubilize the peptide and dilute it to your desired concentration.


• If the overall charge of the peptide is negative, the peptide is acidic. Acidic peptides may be soluble in PBS (pH7.4). If this fails, add a small amount of basic solvent such as 0.1M ammonium bicarbonate to dissolve the peptide and add water to the desired concentration. Peptides containing free cysteines should be dissolved in degassed acidic buffers. The thiol moieties will be rapidly oxidized to disulfides at pH>7.


• If the overall charge of the peptide is zero, the peptide is neutral. Neutral peptides usually dissolve in organic solvents. First, try to add a small amount of acetonitrile, methanol, or isopropanol. For very hydrophobic peptides, try to dissolve the peptide in a small amount of DMSO, and then dilute the solution with water to the desired concentration. For Cys-containing peptides, use DMF instead of DMSO. For peptides that tend to aggregate, add 6 M guanidine.HCl or 8 M urea, and then proceed with the necessary dilutions.

In order to prevent or minimize peptide degradation, store the peptide in lyophilized form at -20°C or preferably at -80°C. If the peptide is in a solution, freeze-thaw cycles should be avoided by freezing individual aliquots.

Positively charged residues: K, R, H, N-terminus
Negatively charged residues: D, E, C-terminus
Hydrophobic uncharged residues: F, I, L, M, V, W, Y
Uncharged residues: G, A, S, T, C, N, Q, P, acetyl, amide

Examples:
RKDEFILGASRHD: (+5) + (-4) = +1 This is considered a basic peptide. See step #2 above.
EKDEFILGASEHR: (+4) + (-5) = -1 This is considered an acidic peptide. See step #3 above.
AKDEFILGASEHR: (+4) + (-4) = 0 This is considered a neutral peptide. See step #4 above.

We cannot predict the solubility of a peptide in water by studying the structure. However, the ε-amino group of Lys and the guanidine function of Arg are usually helpful for estimating solubility, especially for short sequences. In contrast, acidic peptides containing Asp and Glu tend to be insoluble in water, but they can be dissolved easily in diluted ammonia or basic buffers.

Certain basic characteristics can be used to predict solubility:

• Peptides that are shorter than 5 amino acids are usually soluble in aqueous solutions. If the entire sequence consists of hydrophobic amino acids, it will have limited solubility or may even be completely insoluble.
• Hydrophilic peptides containing >25% charged residues (E, D, K, R, and H) and <25% hydrophobic amino acids are usually soluble in aqueous solutions.
• Hydrophobic peptides containing 50% or more hydrophobic residues may be insoluble or only partly soluble in aqueous solutions. It is better to dissolve these peptides in organic solvents such as dimethylsulfoxide (DMSO) if they do not contain C, W or M, dimethylformamide (DMF), acetonitrile, isopropyl alcohol, ethanol, acetic acid, 4?8 M guanidine hydrochloride (GdnHCl), or urea prior to a careful dilution in aqueous solution.
• Hydrophobic peptides containing >75% hydrophobic residues generally do not dissolve in aqueous solutions. Very strong solvents such as TFA and formic acid are required for the initial solubilization. The peptide may precipitate when added to an aqueous buffered solution. High concentration of organic solvent or denaturant may be required to dissolve these peptides.
• Peptides containing a very high proportion (>75%) of D, E, H, K, N, Q, R, S, T, or Y are capable of building intermolecular hydrogen bonds (cross-links) and can thus form gels in concentrated aqueous solutions. These peptides should be dissolved in organic solvents. The initial solvent of choice should be compatible with the experiment. After dissolving the peptides in organic solvent, slowly add (dropwise) the solution to a stirred aqueous buffer solution. If the resulting peptide solution begin to show turbidity, you have reached the limit of solubility.

Crude peptides are not recommended for biological assays. Crude peptides may contain large amounts of non-peptide impurities such as residual solvents, scavengers from cleavage, TFA and other truncated peptides. TFA cannot be totally removed. Peptides are usually delivered as TFA salt. If residual TFA is a problem for your experiment, we recommend other salt forms such as acetate and hydrochloride. These salt forms are usually 20-30% more expensive than the regular TFA salt. This is due to the peptide loss that takes place during the salt conversion and the greater amounts of raw materials required.

>70% purity

• Peptide arrays
• Antigens for antibody production
• Competitive elution chromatography
• ELISA standards for measuring antisera titers

>80% purity

• Western blotting studies (non-quantitative)
• Enzyme-substrate studies (non-quantitative)
• Peptide blocking studies (non-quantitative)
• Affinity purification
• Phosphorylation assays
• Protein electrophoresis applications and immunocytochemistry

>95% purity

• ELISA standards and RIA protocols (quantitative)
• Receptor-ligand interaction studies (quantitative)
• In vitro bioassays and in vivo studies
• Enzyme studies and blocking assays (quantitative)
• NMR studies
• Mass spectrometry
• Other quantitative assays

>98% purity

• SAR Studies
• Clinical trials
• APIs (Active Pharmaceutical Ingredients)
• Commercial products
• X-ray crystallography studies
• Other sensitive experiments: enzyme-substrate studies, receptor-ligand interaction studies, blocking and competition assays

Peptide purity is the amount of the target peptide as determined by HPLC at 214 nm, where the peptide bond absorbs. Water and residual salts are not detected by UV spectrophotometer. Other impurities that can be found in the content include deletion sequences (shorter peptides lacking one or more amino acids of the target sequence), truncated sequences (generated by capping steps to avoid the formation of deletion peptides), and incompletely deprotected sequences (generated during the synthesis or the final cleavage process).

Peptide purity does not include any water or salts in the sample. TFA results from HPLC purification. The free N terminus and other side chains such as Arg, Lys, and His form trifluoroacetates and this allows small amounts of TFA to contaminate the peptides. Peptides are usually delivered as trifluoroacetates containing residual water. Even in lyophilized peptides, varying amounts of noncovalently bound water still exist.

What are other substances (impurities) in the peptides?

Impurities	Non-Purified Peptides	Purified Peptides (HPLC)
Deletion sequences1	✓	✓
Truncation sequences2	✓	✓
Incompletely deprotected sequences3	✓	✓
Sequences modified during cleavage4	✓	✓
DTT (dithiothreitol)	✓
TFA (trifluoroacetic acid)	✓	✓
Acetic acid	✓
Peptides that have undergone side reactions such as proline isomerization or isoaspartimide formation, etc.	✓	✓

The impurities in non-purified peptides are both peptides and non-peptides, the impurities in purified peptides are mostly peptides with modified sequences, except for TFA salt.

• Shorter peptides lacking one or more amino acids of the target sequence.
• Generated by capping steps to avoid the formation of deletion peptides.
• Generated during the synthesis or the final cleavage process.
• Reattachment of protecting groups at other locations on the peptide.

The net peptide content is different from the peptide purity. The net peptide content is the percentage of peptides relative to nonpeptidic materials, mostly counterions 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. Usually, hydrophilic peptides absorb tiny amounts of moisture even under strict lyophilization conditions. Net peptide content may vary from batch to batch depending on the purification and lyophilization processes.

Peptides are usually delivered as TFA salts. If residual TFA would be problematic for your experiment, we recommend other salt forms such as acetate and hydrochloride. These salt forms are usually 20-30% more expensive than the regular TFA salt because of the peptide loss that takes place during the salt conversion and the greater amounts of raw materials required.

Unlike the natural protein synthesis, peptides are synthesized from the C to N terminus. At Biostem, peptide synthesis is performed using Biostem technology based on Fmoc or t-Boc chemistry to protect the alpha amino group. The deprotection agent (piperidine for Fmoc, TFA for Boc) frees the alpha amino group in preparation for coupling the next amino acid in the sequence. This reveals a new N-terminal amine to which the next amino acid may be activated by one of several reagents, forming a peptide bond. When the synthesis is complete, peptides are cleaved from the resin and de-protected. Peptides are then precipitated, washed, and lyophilized.

All materials supplied to Biostem are considered the confidential property of the customer. Biostem provides free HPLC and MS results with your package. Peptides are purified by reverse-phase chromatography. The chromatogram indicates the number and relative amount of by-products. The molecular mass of the peptide is determined by mass spectrometry to confirm that the correct product is being delivered. MS results also show the masses of the main impurities. Additional analysis revealing net peptide content can be performed upon request. Net peptide content is indicated by either amino acid analysis or elemental analysis. These methods allow the verification of the amino acid composition of the peptides. They serve as additional means of confirmation of peptide identity. All synthetic peptides meeting the customer's purity criteria are sent. All residual materials, such as peptides not meeting the customer's purity criteria are discarded. These residual materials can be sent to the customer upon request.

Upon request, Biostem can aliquot part or all of your order into smaller quantities for a minimum fee of $3 per tube. Aliquoted products are more expensive but may save you time, effort and money during determination of peptide solubility. Your peptides will also be more stable because they will not be exposed to as many freeze-thaw cycles, as many openings and closings of the container, mishandling, or bacterial contamination. Peptide oxidation, degradation, and aggregation are less prevalent in aliquoted samples.

APIs (active pharmaceutical ingredients) are the substances in drugs that are pharmaceutically active, such as oxytocin acetate, enfuvirtide acetate, and so on. Catalog peptides are commercially available sequences. They are usually produced in bulk at high levels of purity. These peptides are usually customized to customers' specific requests. For example, specific sequences, modifications, purity levels, or lengths may be required by the customer. The turnaround time for most API peptides is 2-3 weeks.

The minimumal quantity to be ordered should be at leastis 1 mg. At chinapeptide, There there is no maximum upper limit at chinapeptide for research and or GMP peptides.

Biostem has synthesized a peptide of 150 amino acids in length. Peptides of 60 amino acids are synthesized routinely.

Organic reactions are carried out on substrates covalently attached to a polymeric resin. Solid-phase synthesis can be better than the traditional synthesis because the overall reaction takes place much more quickly, the process can be automated with robots, and synthetic intermediates do not need to be isolated because reagents are washed away during each step.

Resin is the polymeric backbone to which substrates are anchored. Different resins have different properties. For example, polystyrene swells in non-polar solvents, while polyethylene glycol swells in polar and non-polar solvents. Linkers are intermediate structures that attack the resin to the substrate. Different linkers can be used to unmask different functional groups on the substrate.

Protecting groups are fragments that binds to functional groups and blocks their reactivity. Some are acid-labile protecting groups such as Boc and tert-Bu ester. Some are base labile protecting groups such as Fmoc and Fm ester. Some others are fluoride-labile protecting groups such as Tmsec and Tmse ester. To ensure specific coupling between the required carboxyl and amino groups, the protecting groups should be easy to attach and remove without changing the rest of the peptide.

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. In this way, these modifications may increase the peptide's biological activity.

Usually, dyes such as biotin and FITC can be introduced either N-terminally or C-terminally. We recommend N-terminus modification for its higher success rate, shorter turnaround time, and ease of operation. Peptides are synthesized from the C-terminus to the N-terminus. N-terminus modification is the last step in the SPPS protocol. No more specific coupling steps are required. In contrast, the C-terminus modification requires additional steps and is usually more complex.
Most dyes are large aromatic molecules. The incorporation of such bulky molecules may help to avoid interactions between the label and the peptide. This will help maintain peptide conformation and biological activity. It is recommended that a flexible spacer such as Ahx (a 6 carbon linker) be included to render the fluorescent label more stable. Otherwise, FITC could easily link to a cysteine thiol moiety or the amino group of lysine at any position.

Peptide Purity is the percentage target sequence amongst the total quantity of peptides. Because peptide bond formation in synthesis is not 100% efficient, not all polypeptide chains are the target sequence. Some chains may not go to completion, or amino acids may not properly bond on certain chains. These deleted sequences make up a certain percentage of peptides in your mixture. We analyze and purify crude peptides using Reverse Phase HPLC in conjunction with Mass Spec Analysis to attain the desired target sequence purity.

After your peptide is purified and lyophilized, the white peptide powder will contain some non-peptide components such as water, absorbed solvents, counter ions and salts. Net peptide content consists of the actual percentage weight of peptide in your final product. This number varies, anywhere from 50 to 90 percent, depending on the purity, sequence and method of synthesis and purification. When calculating the concentration of peptide solution for biological assays or other sensitive peptide experiments, it is essential that you account for peptide content. Peptide concentrations can be determined by subtracting away the non-peptide weight determining the volume of solvent in which to dissolve. For example, when using 1mg of final product to make a 1mg/ml solution of peptide with a content of 80%, you would use 800ul of solvent instead of 1000ul.

Peptide content is not an indication of peptide purity; these are two measurements. Purity is determined by HPLC and indicates the presence/absence of contaminating peptides with undesired sequences. Net peptide content only gives information on the percent of total peptide versus total non-peptide components independently of the presence of multiple peptides. Net peptide content is accurately found by performing amino acid analysis or UV spectrophotometry.

It is difficult to determine the actual peptide concentration based on the weight of the lyophilized peptide. Lyophilized peptides may contain 10-70% water and salts by weight. More hydrophilic peptides generally contain more bound water and salts compared to hydrophobic peptides.

If the peptide has a chromophore in the sequence (W or Y residues), peptide concentration can be conveniently determined based on the extinction coefficient of these residues.

The following steps can be used for the calculations:

• Molar extinction coefficients of chromophoric residues at 280 nm at neutral pH using a 1-cm cell:
• Tryptophan 5560 AU/mmole/ml
• Tyrosine 1200 AU/mmole/ml
• The extinction coefficient of each chromophore in the peptide sequence is generally considered to be additive, that is, the overall molar extinction coefficient of the peptide depends on the types and number of these choromophoric residues in the sequence.
• Calculations: mg peptide per ml = (A280 x DF x MW) / e, where A280 is the actual absorbance of the solution at 280 nm in a 1-cm cell, DF is the dilution factor, MW is the molecular weight of the peptide and e is the molar extinction coefficient of each chromophore at 280 nm
• Hypothetical example: A 50X diluted solution of a peptide with the sequence GRKKRRQRRRPPQQWDCDLYRPYEKT (MW = 3418) reads 0.5 AU at 280 nm in a 1-cm cell.
  To calculate the original peptide concentration in the stock peptide solution: mg peptide/ml = (0.5AU x 50 x 3418 mg/mmole) / [(1 x 5560) + (2 x 1200)] AU/mmole/ml = 10.7
• Cautions:
  
  • Any absorbance calculation assumes that the peptide is unfolded and the chromophores are exposed, which is usually the case in short, soluble peptides. If there are doubts about the solubility or the folding of the peptide, it is advisable to make the measurement under denaturing conditions (e.g., 6M GdnHCl or 8M urea). Obviously, these peptide solutions will be rendered useless, unless the denaturants are removed.
  • If the sequence does not have Trp or Tyr, the only practical option is to do amino acid analysis.

Does your sample contain proteins of interest that are <20 kDa? Please download a protocol on how to detect synthetic peptides using SDS-PAGE, which includes efficient methods for Coomassie blue staining, silver staining, and electroblotting.

Tricine-SDS-PAGE is commonly used to separate proteins in the mass range of 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa.

It is indeed very difficult to see the small peptide by SDS-PAGE. Tris-tricine gel will give you a better resolution. If you just want to detect the peptide, Mass Spec is still the best way to confirm the peptide identity.

Small peptide binds less Coomassie brilliant blue than larger protein. Thus smaller peptides are harder to detect by coomassie staining or silver staining. If you really want to see your peptide on the gel, you can try to load more samples. Changing the gel percentage won't help much unless you think your peptide migrated out of the gel. You can increase the percentage of cross linker in the regular 17% gel. In addition increase the pH of your resolving gel to 9.5 as compared to your regular 8.8. Plus, the addition of urea (4-8M) helps sharpen bands.

If you are going to use western, which is a way more sensitive detection method, please use Western instead of the gel staining. However the peptide may simply pass through the membrane. If you repeat the experiment, try to put two pieces of membrane and shorter time of transfer (less than 1 hour at 200 mA). 0.2um pore could be enough. You can get smaller pore but that shouldn't be necessary. You may want to try semi-dry transfer for 15-20 minutes at the recommended current density (mA/cm2) for the apparatus. A short 15 min transfer time works for most of the small peptides.

If you can plan ahead and synthesize a control small peptide labeled with biotin, you can monitor the transfer process and its ability to bind the membrane with streptavidin-conjugated HRP.

Dimethyl sulfoxide (DMSO) is an organosulfur compound with the formula (CH3)2SO. DMSO is frequently used in cell banking applications as a cryoprotectant. DMSO prevents intracellular and extracellular crystals from forming in cells during the freezing process. For most cryopreservation applications, DMSO is used at 10% concentration and is usually combined with saline or serum albumin.

Hydrophobic peptides can be easily dissolved in DMSO. However peptides in DMSO could be cytotoxic to the cells although DMSO increases cell permeability. High concentration of DMSO should never be used for cell culture. 5% is very high and will be dissolving the cell membranes. Most cell lines can tolerate 0.5% DMSO and some cells can tolerate up to 1% without severe cytotoxicity. However the primary cell cultures are far more sensitive. So if it is primary cell you are using then do a further dose/response curve (viability) at concentrations below 0.1%.

So for very hydrophobic peptides, try to dissolve the peptide in a small amount of DMSO (30-50ul, 100%), and then slowly add (dropwise) the solution to a stirred aqueous buffer solution like PBS or your desired buffer to the desired concentration. If the resulting peptide solution begin to show turbidity, you have reached the limit of solubility. Sonication will help dissolving the peptides.

Rule of thumb:

• 0.1% DMSO is considered to be safe for almost all cells.
• 0.5% DMSO as the final concentration has been used widely for cell culture without cytotoxicity.
• 1% DMSO doesn't cause any toxicity to some cells but 0.5% DMSO is recommended.
• 5% DMSO was used successfully for some cells.
• To keep the final concentration to 0.5%, you can make 200x stock in 100% DMSO.