Q
How to preserve the synthesized peptides?
Peptides generally need to be stored away from light for long-term storage, and should be stored at -20 degrees, and can be stored at 4 degrees for short-term. Can be shipped at room temperature for short periods of time. Peptides are stable at -20°C, especially when freeze-dried and stored in a desiccator. Freeze-dried peptides can be kept at room temperature before exposing them to air. This will reduce the effect of humidity, and when freeze drying is not possible, the best approach is to store small working samples. For peptides containing Cys, Met or TrP, deoxygenation buffer is essential for their dissolution, because this peptide can be easily oxidized by air, and nitrogen or hydrogen gas slowly flowing through the peptide before sealing the bottle will also reduce the oxidation. Peptides containing GIn or Asn are also prone to degradation, and all these peptides have a limited lifetime compared to those without these problematic simplicity.
Q
If my peptide is 95% pure, what is the other 5%?
Peptide purity is usually determined by HPLC using a gradient of 1% acetonitrile per minute. During the synthesis process, the cross-linking efficiency between amino acids cannot always reach 100%, thus producing a series of impurities with missing amino acids. Most of these amino acid-deleted impurities were removed during purification, but a few had chromatographic profiles that closely resembled the target peptide. Contaminant peptides with these amino acid deletions remaining in the peptide sample make up the remaining few percent.
Q
How are peptides purified?
Peptide purification generally uses a reversed-phase column (such as C8, C18, etc.), 214nm. The buffer system is usually a solvent containing TFA, pH 2.0. Buffer A contains 0.1% TFA in H2O, and Buffer B contains 1% TFA/ACN/pH2.0. Dissolve with Buffer A before purification; if the dissolution is not good, dissolve with Buffer B and then dilute with Buffer A; for highly hydrophobic peptides, sometimes a small amount of Formic Acid or acetic acid needs to be added. HPLC analysis of crude peptide products, if the peptide is not long (below 15aa), there will generally be a main peak, and the main peak is usually the full-length product; for long peptides over 20aa, if there is no main peak, HPLC needs to be used with Mass to determine the molecular weight, and then determine which peak is the peptide to be synthesized.
Q
How should the ends of the peptide be handled? Keep it free or block it?
Peptides are used to simulate proteins. In order to mimic the performance of proteins, we need to synthesize polypeptides with similar structures and charges 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 gene body protein. We need to change the compositing strategy to make them consistent. In general: If the sequence is from the C-terminus of the protein, shield the N-terminus by acetylation; if it is a sequence from the N-terminus of the protein, shield the C-terminus by amidation; if it is from the middle part of the protein, use acetylation and amidation to shield the Both ends are shielded.
Q
What are the advantages of PEG-modified peptides?
Polyethylene glycol modification is to add high molecular polymer (ethylene glycol) to the target molecule through covalent bonding. Polyethylene glycol modification deceives the host cell's immune system by camouflaging the polypeptide, enhances the therapeutic effect of the polypeptide, and increases the solubility and bioavailability of hydrophobic drugs. It may also prolong the circulation of peptides by reducing renal clearance.
Q
What should be paid attention to when introducing fluorescent modification into peptides?
KS-V peptide suggests adding a linker between the polypeptide molecule and the fluorescent modification, which can reduce the impact of the fluorescent modification on the folding of the polypeptide and the binding to the receptor. However, if the purpose of fluorescence modification is to quantify the fluorescence migration between different structures, it is not recommended to introduce Linker.
Q
What should be paid attention to when designing phosphorylation-modified peptides?
KS-V peptide recommends that when designing phosphorylation modifications, the phosphorylation modification should not exceed 10 amino acids from the N-terminus to avoid a decrease in coupling efficiency.
Q
Can you do D-peptide? How many types of D-type natural amino acids are there?
Can. There are 19 kinds of D-type natural amino acids. Gly in natural amino acids has no chiral structure, and the D-type structure of other natural amino acids can be selected in Keshengjing peptide. For details, please log on to the official website to view the list of special amino acids.
Q
How many phosphorylation sites can be included in KS-V peptide synthesis?
Generally, there are at most three, and it needs to be analyzed according to the specific sequence.
Q
Why do polypeptides undergo N-terminal acetylation and C-terminal amidation modification?
Such modifications can confer on the polypeptide sequence the properties of the native protein.