0 1
How should I handle and preserve peptides?
Polypeptide in the form of freeze-dried powder can be transported stably at room temperature after sealed packaging, and the dissolved polypeptide should not be stored for a long time.
Guidelines for the preservation of Antrolex peptides: Polypeptides that need to be preserved for a long time should be stored in a sealed container filled with desiccant in the form of freeze-dried powder, and stored at -20℃, and the effect is better at -80℃, which can avoid the degradation of polypeptides to the greatest extent. This preservation method can preserve the polypeptide for several years, avoiding degradation and oxidation by bacteria and avoiding the formation of secondary structure.
Unpacking: Before unpacking and weighing, please balance the polypeptide to room temperature in the dryer. Because peptides are often hygroscopic, peptides that are unbalanced at room temperature are easy to coagulate after opening the lid, thus reducing the stability of polypeptide products.
Weighing: quickly weigh the polypeptide you need and store the remaining polypeptide at-20 C or lower. Compared with other peptides, peptides containing cysteine, methionine, tryptophan, asparagine, glutamine and glutamic acid have shorter shelf life.
02
How does polypeptide dissolve?
The solubility of the polypeptide produced by Antrolex depends largely on the polarity of the polypeptide. Acidic protein is soluble in alkaline solution, while alkaline protein is soluble in acidic solution. Hydrophobic peptides and neutral peptides containing a large number of nonpolar 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). Polypeptides containing methionine or cysteine cannot be dissolved in DMSO because DMSO may lead to side chain oxidation.
Dissolution test of polypeptide: Take a small part of polypeptide for dissolution test before dissolution. You need to test several different solvents until you find the most suitable one. Ultrasonic treatment helps to break up particles and increase solubility. (Note: Ultrasonic treatment will cause solution heating and polypeptide degradation. )
Assign a value of-1 to each acidic amino acid, including aspartic acid (d), glutamic acid (e) and carboxyl terminal -COOH. The value of each basic amino acid is+1, including arginine (r), lysine (k), histidine (h) and amino-terminal -NH2. Then calculate the charge number of the whole polypeptide.
If the charge of the whole peptide is positive, it means that the peptide is alkaline. Try to dissolve it with distilled water first; If it is insoluble in water, try to dissolve it with a small amount of 10%-25% acetic acid. If it still fails, add some TFA( 10-50 microliters) to dissolve it, and then dilute it with water to the ideal concentration.
If the charge of the whole peptide is negative, it means that the peptide is acidic. Acidic peptides can be dissolved by 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 it to the ideal concentration. Polypeptides containing free cysteine should be dissolved in degassed acidic buffer, because sulfhydryl groups will be rapidly oxidized to disulfide when the PH value is greater than 7.
If the charge of the whole peptide is zero, it means that the peptide is neutral. Neutral peptides are usually soluble in organic solvents. First, Antrolex suggests that you try adding a small amount of acetonitrile, methanol or isopropanol. For highly hydrophobic peptides, a small amount of dimethyl sulfoxide can be dissolved and then diluted to the ideal concentration with water. For peptides containing free cysteine, DMF should be used instead of DMSO. For peptides with aggregation tendency, 6M guanidine hydrochloride or 8M urea can be added and then diluted as necessary.
In order to prevent or minimize polypeptide degradation, please store the polypeptide in the form of freeze-dried powder at -20℃, preferably -80℃ ... If the solution peptide needs to be preserved, it is best to keep it in a small sample to avoid repeated freezing and thawing. Samples that are not used up after thawing should be thrown away. Bacterial degradation sometimes becomes a problem for peptide solution, so please dissolve the peptide in sterile water or peptide solution and filter for sterilization.
Basic amino acids: K, R, H, N terminal
Acidic amino acids: D, E, C terminal
Polar neutral amino acids: F, I, L, M, V, W, Y.
Nonpolar hydrophobic amino acids: g, a, s, t, c, n, q, p, acetyl, amido.
For example:
Rkdefilgasrhd: (+5)+(-4) =+1is considered as an alkaline polypeptide.
Ekdefilgasehr: (+4)+(-5) =-1is considered as an acidic polypeptide.
AKDEFILGASEHR:(+4)+(-4) = 0 is considered as a neutral polypeptide.
03
Can you predict whether a peptide is soluble?
Antrolex can't predict the solubility of polypeptide in water by studying its structure. The ε -amino group of lysine and guanidine group of arginine are usually helpful to predict solubility, especially short peptides. On the contrary, acidic peptides containing aspartic acid and glutamic acid are often insoluble in water, but soluble in dilute ammonia or alkaline buffer.
04
How to choose the purity of peptides suitable for your own research?
Antrolex does not recommend the use of crude peptides in biological experiments. Crude peptides may contain a lot of non-peptide impurities, such as residual organic solvents, scavengers, TFA and other incomplete peptides. TFA cannot be completely eliminated, and the delivered peptide usually exists in the form of TFA salt. If the residual TFA affects your experiment, we suggest that you use other salt forms, such as acetate and hydrochloride. These salts are usually more than 20-30% more expensive than traditional TFA salts. This is because more peptides are lost in the transformation process and more raw materials are needed.
Antrolex recommends the following levels of peptide purity for various projects:
& gt70% peptide purity
Peptide microarray
As an antigen for preparing antibodies
chromatography
Detection of antiserum titer by enzyme-linked immunosorbent assay
& gt80% peptide purity
Protein blot (non-quantitative)
Enzyme substrate peptide (non-quantitative)
Blocked peptide (non-quantitative)
Affinity purification
Phosphorylation detection
Application of protein Electrophoresis and Immunocytochemistry
& gt95% peptide purity
Standard enzyme-linked immunosorbent assay and RIA (quantitative)
Receptor ligand interaction (quantitative)
In vivo and in vitro
biological assay
Enzyme research and blocking experiment (quantitative)
Nuclear magnetic resonance study
mass spectrometric analysis
Other quantitative detection
& gt98% peptide purity
Research on synthetic aperture radar
clinical trial
Raw materials (active ingredients)
industrial product
Study on x-ray crystal
Other sensitive experiments: interaction, blocking and competition experiments between enzyme and substrate, receptor and ligand.
05
What is the purity of polypeptide?
The purity of polypeptide refers to the content of target polypeptide detected by HPLC at 2 14nm (2 14nm is the absorption wavelength of peptide chain), and water and residual salt cannot be detected by ultraviolet spectrophotometer. Other impurities that can be found include: deleted sequences (target sequences with one or more amino acid residues deleted), truncated sequences (sequences generated during capping) and deprotected incomplete sequences (sequences generated during the whole synthesis process or the final cleavage process).
The purification of polypeptide does not involve water and salt. HPLC purification will produce a small amount of TFA, for example, free amino terminal and other side chains such as Arg, Lys and His will produce a small amount of TFA impurities. Peptides usually delivered by Antrolex contain trace TFA and residual water. Even in the freeze-dried state, due to the different binding ability of * * *, water will exist in different degrees.
The impurities contained in the polypeptide before purification include polypeptides and non-polypeptide substances, and the impurities contained in the purified polypeptide are mostly peptides with modified sequences except TFA salts.
A target sequence with one or more amino acid residues deleted.
A capping operation to avoid the generation of deleted sequences, in which truncated sequences will be generated.
It is produced in the whole synthesis process or the final cracking process.
The protecting group is reconnected to other positions of the polypeptide.
06
What is the net peptide content?
The net content of peptide is different from the purity of polypeptide. Net peptide content refers to the amount of polypeptide compared with non-peptide substances (mainly counter ions and water). The net peptide content can be determined by amino acid analysis. Generally speaking, hydrophilic peptides will absorb a small amount of water even in a strictly freeze-dried state. Due to the purification and freeze-drying process, the net content of peptides in different batches will be different.
07
What about synthetic peptides?
Different from the synthesis of natural protein, the direction of artificial synthesis is from C end to N end. Antrolex's polypeptide synthesis is based on the platform of PeptideSyn technology, namely the Fmoc or t-Boc method. The specific synthesis consists of the following cycles: ① Deprotection: piperidine must be used to remove the protective groups of amino groups on Fmoc-protected columns and monomers. ② Activation crosslinking: the carboxyl group of the next amino acid is activated by the activator. The activated monomers react with free amino groups, cross-link and form peptide bonds. ③ Cycle: Repeat these two reactions until the synthesis is completed. Then the synthesized peptide was cut and deprotected from the resin. Finally precipitate, eluting and freeze-dry.
08
What salt form should I use?
Peptides are usually delivered in the form of TFA salts. If the residual TFA is problematic for your experiment, we suggest using other salt forms, such as acetate and hydrochloride. These salt forms are usually 20-30% more expensive than common TFA salts, because peptide loss occurs during salt conversion and more raw materials are needed.
09
How to check the quality of synthetic peptides?
The company keeps all information provided by customers strictly confidential. Antrolex provides free HPLC and MS test results when sending products. All the peptides in the company are purified by reversed-phase chromatography. The molecular weight of peptide is determined by mass spectrometry to determine whether the product is correct, and the results of MS detection can also show most of the main impurities. If necessary, it can also provide peptide net content detection, such as amino acid analysis or elemental analysis. These methods can confirm the amino acid composition of polypeptide and can be used as supplementary methods for polypeptide confirmation. All delivered peptides have reached the purity required by customers. Those peptides that do not meet the purity requirements are discarded. Of course, if customers need it, they can also send it to them.
10
Does the company provide packaging services for synthetic peptides?
Upon request, Antrolex can divide some or all of your orders into small parts for free. Because a small amount of packaging can avoid repeated freezing and thawing, reduce the number of container opening and closing, reduce the chance of improper handling or bacterial contamination, and make your polypeptide more stable.
Welcome to ask questions. If you have any unclear questions, please feel free to contact us!