FAQ
| Peptide Synthesis: Handling and Storage of Synthetic Peptides | |||||||||||||||||||||||||||
1. How should I store and handle my synthesized peptides?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.
2. How should I dissolve 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.
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 Examples:
3. Can I predict whether a peptide will be soluble?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:
4. How do I choose the best level of peptide purity for my research?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
>80% purity
>95% purity
>98% purity
5. What is peptide purity?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?
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.
6. What is net peptide content?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.
7.What salt form should I use?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.
8. How are peptides synthesized?Unlike the natural protein synthesis, peptides are synthesized from the C to N terminus. At chinapeptide®, peptide synthesis is performed using PeptideSyn 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.
9. What are your QC standards for peptide synthesis?All materials supplied to chinapeptide are considered the confidential property of the customer. chinapeptide 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.
10. Can you aliquot my peptides?Upon request, chinapeptide 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.
11. What are APIs, catalog peptides and custom peptide synthesis?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.
12.What is the minimum quantity for one order?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.
13. What is the maximum peptide length that chinapeptide can produce?chinapeptides has synthesized a peptide of 120 amino acids in length. Peptides of 50 amino acids are synthesized routinely.
14. What is solid-phase synthesis?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.
15. What are resins and linkers?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.
16. What is a protecting group?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.
17. What are acetylation and amidation?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.
18. Is a spacer required for fluorescent modification?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.
19. How to calculate the peptide concentration?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:
20. How to Detect Small Peptides by SDS-PAGE?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.
21. How to dissolve peptides in DMSO?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:
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