Frequently Asked Questions about Peptide
1. If the peptide is in the form of freeze-dried powder, it can be transported stably under normal temperature conditions through a sealed package.
2. Peptides in the dissolved state should not be stored for a long time.
3. Peptides that need to be stored for a long time should be stored in the form of freeze-dried powder in a sealed container containing a desiccant and stored at -20°C. The effect is better at -80°C, which can minimize peptide degradation. This storage method allows the peptide to be stored for several years, avoiding bacterial degradation and oxidation, and also avoiding the formation of secondary structure.
The solubility of a polypeptide largely depends on the polarity of the polypeptide. Acidic proteins are dissolved in alkaline solutions, while basic proteins can be dissolved in acidic solutions. Hydrophobic and neutral polypeptides containing a large number of uncharged polar amino acid residues or hydrophobic amino acids can be dissolved in a small amount of organic solvents. , Such as DMSO, DMF, acetic acid, acetonitrile, methanol, propanol or isopropanol, and then diluted with water (distilled water). Peptides containing methionine or cysteine cannot be dissolved with DMSO, because DMSO may cause side chain oxidation.
Peptide dissolution test: Before dissolving the peptide, take a small part of the peptide dissolution test. You need to test several different solvents until you find the most suitable one (determined according to experimental requirements and solubility experiments). Ultrasound treatment helps to break up the particles and increase solubility. (Note: Ultrasonic treatment will cause heating of the solution and degradation of peptides.)
1. Assign a value of -1 to each acidic amino acid, including aspartic acid (D), glutamic acid (E), and C-terminal -COOH. Each basic amino acid is assigned a value of +1, including arginine (R), lysine (K), histidine (H), and N-terminal -NH2. Then calculate the charge of the entire polypeptide.
2. If the entire peptide is positively charged, it means that the peptide is basic. You can first try to dissolve with distilled water; if it is insoluble in water, then try to dissolve with a small amount of 10%-25% acetic acid, if it still fails, add some TFA (10-50 microliters) to solubilize, and then dilute with water to the desired concentration .
3. If the entire peptide is negatively charged, it means that the peptide is acidic. You can try to dissolve with distilled water first; if it is insoluble in water, you can try to dissolve with PBS (pH 7.4), if it is insoluble, add a small amount of alkaline solvent, such as 0.1 M ammonium bicarbonate, and then add water to dilute to the desired concentration . Peptides containing free cysteine should be dissolved in a degassed acid buffer, because when the pH is greater than 7, sulfhydryl groups will be rapidly oxidized to disulfides.
4. If the charge of the entire peptide is zero, the peptide is neutral. Neutral peptides are usually soluble in organic solvents. First, try adding a small amount of acetonitrile, methanol, or isopropanol. For highly hydrophobic peptides, use a small amount of dimethyl sulfoxide to dissolve, and then dilute with water to the desired concentration. For peptides containing free cysteine, use DMF instead of DMSO. For peptides that tend to aggregate, 6M guanidine hydrochloride or 8M urea can be added, and then the necessary dilutions are made.
In order to prevent or minimize the degradation of peptides, please store the peptides in the form of freeze-dried powder at -20°C, preferably -80°C. If you need to preserve the solution peptides, it is best to store them in small samples to avoid repeated freezing and thawing. If a sample is not used up after thawing, it should be thrown away. Bacterial degradation can sometimes become a troublesome solution for peptides, so please dissolve peptides in sterile water or peptide solutions to filter and sterilize.
Most peptides are dissolved in sterile distilled water. When dissolving for the first time, make sure that the initial concentration is greater than the required concentration. If the peptide has only limited solubility, this allows the addition of other dissolving agents or buffer salts.
If the solubility of the peptide in water is limited, there are several options to help dissolve it:
Use dilute acetic acid for basic peptides (including Arg, Lys, His)
Dilute ammonia water for acidic peptides (including Asp, Glu)
10% organic modifiers (acetonitrile, Methanol) for extremely hydrophobic peptides
DM50 or DMF for extremely insoluble peptides
Concentrated solutions of guanidine hydrochloride or urea are also very useful, combined with the above methods, sonication is also an effective means to dissolve peptides.
Peptides are very stable at -20°C, especially freeze-dried and stored in a desiccator. Before exposing them to air, freeze-dried peptides can be placed at room temperature. This will reduce the influence of humidity. When freeze-drying is not possible, the best method is to store it in a small amount of work.
For peptides containing Cys, Met orTrP, deoxygenating buffer is essential for their dissolution, because this peptide can be easily oxidized in the air. Before sealing the bottle, nitrogen or argon flowing through the peptide will also reduce the oxidation. Peptides containing Gln or Asn are also prone to degradation, and all these peptides have a limited life span compared to those without these problematic glycosides.
Accurate determination of peptide concentration is more complicated than many people expected. In fact, there is no simple general method that can be used. Peptide weighing method and UV absorption are the two most commonly used methods.
1. Peptide weighing method It can only provide a rough estimate of the peptide content. First of all, peptide powder is not easy to dispose of. Accurately weighing small amounts (<3 mg) is particularly problematic. Another problem is that peptides can contain various levels of water and relatively low levels of counter ions even after being lyophilized on a large scale.
The water content ranges from about 5% to 20%, and some even> 40%. The actual content depends on the peptide sequence. The type and quantity of counterions depend on the solvent used for peptide purification and the peptide sequence. The uncertainty generated by these water molecules cannot be neglected.
2. Polypeptide UV absorption. If your peptide contains a tyrosine or tryptophan residue, the quantitative analysis of the peptide becomes easier. The molar absorbance of tryptophan at 282 nm [1/(M*cm)] is 5700, and the molar absorbance of tyrosine at 275 nm is 1400.
In 0.1N NaOH, the -OH group of tyrosine is fully deprotonated. This makes the absorption peak of tyrosine 293 nm. Under this condition, its molar absorbance increased to 2400. It is worth noting that if the side chains of the peptide interact with each other, the UV absorption rate can be different. Regarding small hydrophilic peptides, this is not a problem.
When polypeptides are in an aqueous solution, these peptides usually adopt a stretched conformation, and all polypeptide side chains are completely exposed to the solvent. But regarding long hydrophobic peptides, this assumption is incomplete and correct. Sometimes low-level aggregation and folding can be observed. For this reason, we believe that the absorbance of tyrosine at 293 nm in 0.1N NaOH is the best, because under this condition, the peptide is completely denatured. Its only drawback is that the sample used for concentration determination cannot be recovered.
If your peptide does not contain tryptophan or tyrosine, an accurate understanding of the peptide concentration is crucial. The only reasonable choice is amino acid quantitative analysis. Of course, this is beyond the daily operations of most laboratories.
Based on the above discussion, in order to accurately determine the concentration of the peptide, we strongly recommend adding a tyrosine residue to the N- or C-terminus of your peptide.
Remetide Biotech, LLC is a manufacturer specializing in the production of high-purity, high-quality protein peptides. We have a laboratory factory in China, and our colleagues have offices in the United States and Hong Kong.
We provide custom synthesis and R&D services for protein peptides. Welcome your inquiry: 19498788363 (Mr. Yao)
North America: Remetide 18701 Arenth Ave, City of Industry, CA 91748
Asia Pacific & Europe: Peptide HK Room E02, Blk. E, 9/Floor Kwai Shing Ind. Bldg., Tai Lin Pai Rd., Kwai Chung Hong Kong
Choosing an excellent supplier plays a crucial role in the quality of later peptide synthesis.
1. When the company was established. For companies that have been established for a short time, it is recommended to choose carefully, because the systems of such companies are not mature, especially the control of quality.
2. Manufacturers with their own factories will be more professional. Many peptide synthesis companies use the form of technology outsourcing. Generally, peptide manufacturers with their own factories are better in terms of production and technical stability.
3. Companies specializing in peptide synthesis services are better. Some biological companies only do peptide synthesis incidentally. Such companies often lack professional laboratories and production workshops. If the team is not professional, it is difficult to synthesize difficult peptides.
4. Manufacturers whose prices are too low should choose carefully. Many peptide synthesis service companies attract customers with low prices at first, and then save costs in terms of raw materials, purity, process, etc., so it is difficult to produce high-quality products.
5. A company with complete qualifications. Manufacturers who have been filed by relevant U.S. departments or implement relevant regulations will have complete production qualifications, and such suppliers will be more professional and subject to stricter supervision.
Polypeptide solid-phase synthesis is a major technological breakthrough in peptide synthesis. Not only does she not have to purify intermediates, but the synthesis process can be carried out continuously, thus laying the technical foundation for peptide synthesis.
The current mainstream peptide synthesis method is solid-phase synthesis. The basic process is based on Fmoc chemical synthesis,
1: The carboxyl group of the C-terminal amino acid of the target polypeptide to be synthesized is connected to an insoluble polymer resin in the form of a covalent bond
2: The amino group of this amino acid is used as the starting point of polypeptide synthesis, and the carboxyl group of its amino acid acts at the same time to form a peptide bond
3: Repeat this process continuously, and then you can get the target peptide.
The peptides provided by Remetide are in powder form, generally gray-white, and the color of the peptide powder varies with different compositions.
The preservation and storage of peptides generally require protection from light. Long-term storage should be stored at -20 degrees, and short-term storage can be stored at 4 degrees. Can be shipped at room temperature for a short time.
First of all, it is clear that in theory, the purity of the drug polypeptide is as high as possible.
The most common purity that people use is >95% (HPLC). This purity is suitable for most applications. As an antigenic peptide, purity >85% is sufficient. If it is used for NMR and crystallization, the purity should be >98%, the higher the better. If a large number of peptides are used for screening, the crude product can be used (50%). After all, the higher the purity, the higher the cost.
Special attention should be paid to the fact that synthetic peptides contain a small amount of TFA: after all, peptides are chemically synthesized and contain a small amount of organic reagents. These reagents are cytotoxic, so removal of TFA should be required if these peptides are to come into contact with cultured cells, or if the peptides are required to have some activity
So how to choose the appropriate purity, the customer needs to judge according to the specific situation of the desired drug peptide.