Q: what should be done with the ends of the polypeptide? Is it free or shielded?
A: peptides are used to mimic proteins. In order to mimic the performance of proteins, we need to synthesize peptides with similar structure and charge to proteins. When a peptide is “cut out” from a protein, the number of charges at both ends will be different from that of the body protein.
We need to change the synthesis strategy to make them consistent. In general: if it comes from the C-terminal sequence of protein, N-terminal is shielded by acetylation; if it is from N-terminal sequence of protein, C-terminal is shielded by amidation; if it is from the middle part of protein, both ends are shielded by acetylation and amidation.
Q: if my peptide purity is 95%, what are the remaining 5%?
A: the purity of peptides is usually determined by HPLC with a standard acetonitrile gradient of 1% per minute. In the process of synthesis, the cross-linking efficiency of amino acids is not always 100%, so a series of amino acid missing impurities are produced.
Most of these amino acid deficient impurities were removed in the purification process, but a few impurities had similar chromatographic performance to the target peptide. These amino acid missing impurity peptides remain in the polypeptide sample and constitute the remaining several percentage points.
Q: how to re dissolve peptides?
A: the dissolution of polypeptide is related to the sequence of polypeptide. First try distilled water and ultrasound (1-10mg / ml). If not, calculate the number of basic residues (Lys, Arg, his and free amino terminal) and acid residues (Gly, ASP and free carboxyl end) in the peptide.
If there are too many basic groups, 1n acetic acid can be added until it is dissolved. If there are many acid groups, add 1n ammonia until it is dissolved.
If a buffer solution is needed in the experiment, it is recommended not to add salt before the peptide is completely dissolved, because salt will reduce the solubility of peptide. If the peptides in the above solutions are insoluble, try a small amount of * * such as DMSO, acetonitrile or * *.
Q: what is the net content of polypeptide and what is its significance?
A: the weight of dried polypeptides includes not only polypeptides, but also some non peptide components, such as water, absorbed solvents, coordination ions and salts. The net content of peptide refers to the weight percentage of peptide in it.
The value range of this percentage is very large, which may range from 50% to 90%. It depends on the purity, sequence and synthesis and purification methods. Do not confuse the net content of peptide with the purity of peptide.
They are two completely different concepts. The purity is usually determined by HPLC. Purity is defined as the percentage of components with correct sequence in peptide sample, while the net content of peptide refers to the percentage of peptide material relative to non peptide substance in the sample.
The net content of peptide is usually determined by amino acid composition analysis or ultraviolet spectrophotometry. This information is mainly used in some experiments which are sensitive to the concentration of peptide, and it is very important to calculate the concentration of peptide.
Q: how to choose the purity of peptides for different experiments?
A: the purity of peptide is a very important indicator, and the choice of purity depends on the purpose of the experiment. For the less sensitive screening test, it is recommended to use crude product or > 75%, and for the grade of * *, it is recommended to use > 85%.
For the study of the interaction between receptor and ligand, biological assay research, or cell level research, it is recommended that > 95% and > 98% for the structural study.
Q: how to preserve peptides?
A: if possible, the polypeptides should be preserved in the form of lyophilized powder at – 20 ℃. In this way, the degradation of * *, the formation of secondary structure, oxidation or other modification can be avoided in a few years.
The stability of polypeptide in solution is poor, so it is strongly recommended to use sterilized water or sterilized filter for dissolution. If there are Cys, met or Trp residues in the sequence, they should be dissolved in anaerobic solvent to avoid oxidation.
When using frozen solution, it is recommended to pack the solution separately to avoid the damage to polypeptide caused by repeated dissolution and freezing.
The recommended range of pH is 3-6. Before use, it is necessary to ensure that the packaging container and the contents are returned to room temperature to avoid water absorption.
When the peptide solution is ready to use, keep the peptide solution at low temperature but not frozen during use. Long term storage (3 months to 5 years): lyophilized powder, freeze-dried at – 20 ℃; medium-term storage (0-3 months): refrigerated liquid or lyophilized powder at – 20 ℃; short-term storage (< 1 week): refrigerated liquid or lyophilized powder.
Q: what is the common way to produce peptides?
A: the linear peptide chain is extended by Fmoc solid-phase synthesis method, and amino acids are connected gradually from C-terminal to N-terminal. In the beginning, two amino acids were connected to the insoluble support resin through an acid sensitive linker.
The second Fmoc protected amino acid was linked after the Fmoc protecting group was removed by using the method of pre activation or “one pot cooking”. After the target sequence was linked, the peptide chain was eluted from the resin with TFA to obtain the crude product.
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