Q: How do you dissolve peptides?

A: We recommend a small test with distilled or sterile water before dissolution. Short oligopeptides (less than 5aa) can often be dissolved directly.

For other peptides, it is often necessary to optimize dissolution conditions based on the sequence of the peptide and test several different solvents until an optimal method is found (ultrasonic treatment helps to break up particles and increase solubility).

The polarity of the peptide has a strong effect on the solubility: acidic proteins are more likely to dissolve in alkaline solutions, while acidic solutions are more likely to dissolve in basic proteins.

You can use the following table

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According to the above principles, a score greater than 0 is an alkaline peptide, a score less than 0 is an acidic peptide, and a score of 0 is a neutral peptide.

For example:

RKDEFILGASRHD: (+5) + (-4) = +1 is considered to be an alkaline polypeptide

Ekdefilgasehr: (+4) + (-5) = -1 is considered an acid polypeptide

AkdeFilgasehr: (+4) + (-4) = 0 is considered a neutral polypeptide

1. For alkaline peptides, if pure water cannot be dissolved, try 10%-30% acetic acid;

If the peptide is still insoluble, test pure acetic acid and trifluoroacetic acid TFA(<

50μl) to dissolve, and then dilute the peptide solution to desired concentration.

2. For acidic peptides, if pure water does not dissolve, try 13% ammonia (v/v) to dissolve and dilute to desired concentration.

If the polypeptide sequence contains cysteine Cys (C), which cannot be dissolved with ammonia, dimethylformamide (DMF) or N-methylpyrrolidone (NMP) should be used for dissolution.

For peptides that are easy to aggregate, 6M guanidine hydrochloride (guanidine?

HCl or 8M of Urea to dissolve and then dilute to the desired concentration.

3. Neutral peptides should be dissolved in machine solvent.

First, try to use acetonitrile, or methanol or isopropanol for dissolution;

For very hydrophobic polypeptides, a small amount of dimethyl sulfoxide (DMSO) is first dissolved and diluted to the desired concentration with water.

Similarly, if the polypeptide sequence contains cysteine Cys (C), it is dissolved with dimethylformamide (DMF) or N-methylpyrrolidone (NMP).

For peptides that are easy to aggregate, 6M guanidine hydrochloride (guanidine?

HCl or 8M of Urea to dissolve and then dilute to the desired concentration.


Q: How do you preserve peptides?

A: 1. To facilitate storage and subsequent use, it is recommended to dissolve the polypeptide to a concentration of about 1-2 mg/ mL.

2. To prevent or minimize peptide degradation, store peptides in freeze-dried form at -20°C, -80°C is preferable.

If the solution peptides need to be preserved, it is best to store them in small samples to avoid repeated freezing and thawing.

If a sample is not used after thawing, it should be thrown away.

Bacterial degradation can sometimes be a problem for peptides in solution, so please dissolve the peptides in sterile water or in a peptide solution for filtration and sterilization.

3. Polypeptide sequences containing methionine Met (M), cysteine Cys (C) or tyrosine Tyr (Y) are recommended to be stored in an oxygen-free environment to prevent oxidation.


Q: How do I choose the right peptide purity for my research?

A: Crude peptides are not recommended for use in biological experiments.

Crude peptides may contain large amounts of non-peptide impurities, such as residual organic solvents, scavengers, TFA, and other incomplete peptides.

TFA cannot be completely eliminated and usually delivered peptides are present in the form of TFA salts.

If residual TFA affects your experiment, we recommend other salt forms, such as acetate and hydrochloride.

These salts are usually 20-30% more expensive than conventional TFA salts.

This is due to more peptide loss during conversion and the need for more raw materials.

Novopro recommends the following levels of peptide purity for various items:

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Q: How to dispose of and preserve peptides after receiving them?

Answer: the polypeptides in the form of lyophilized powder can be transported stably at room temperature through sealed packaging, while the polypeptides in the dissolved state should not be stored for a long time.

Peptides preservation guidelines: peptides that need to be stored for a long time should be stored in the form of lyophilized powder in a sealed container containing desiccant, stored at -20℃, -80℃ better effect, can avoid the degradation of the peptide to the maximum extent.

This storage allows peptides to be stored for several years, avoiding degradation and oxidation by bacteria, and avoiding the formation of secondary structures.

Unpacking: Before unpacking and weighing, balance the peptides in a dryer to room temperature.

Because peptides often have hygroscopicity, peptides that have not been balanced to room temperature are easy to coagulate after opening the lid, thus reducing the stability of the peptide products.

Weighing: quickly weigh the peptides you need and store the remaining peptides at -20°C or lower.

Compared with other peptides, peptides containing cysteine, methionine, tryptophan, asparagine, glutamine, and the N-terminal of glutamate have a shorter shelf life.


Q: What is the purity of a polypeptide?

A: The purity of the peptide is the amount of the target peptide detected by the HPLC method at 214nm (the absorption wavelength of the peptide chain). The UV spectrophotometer cannot detect water and residual salts.

However, other impurities found include deletions (target sequences that are missing one or more amino acid residues), truncated sequences (sequences produced during caping) and unprotected incomplete sequences (sequences produced during the entire synthesis or final cleavage).

Peptide purification does not involve water or salt.

Purification by HPLC will produce a small amount of TFA, such as free amino terminal and other side chains such as Arg, Lys, His can produce a small amount of TFA impurities.

Usually delivered peptides contain trace amounts of TFA and residual water.

Even in the lyophilized state, water exists to varying degrees depending on the ability of covalent binding.

What other substances (impurities) are present in the polypeptide?

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The impurities contained in the peptides before purification include peptides and non-peptides. The impurities contained in the peptides after purification are mostly peptides with modified sequences except TFA salts.

1. Target sequence with one or more amino acid residues missing.

2. The capping operation is carried out to avoid the generation of missing sequence, and the truncated sequence is generated in the capping process.

3. It is produced during the whole synthesis process or the final pyrolysis process.

4. Protective groups are reattached to other sites of the peptide.


Q: how does gootuo conduct quality control on synthetic peptides?

A: All synthetic peptides are analyzed by HPLC and MS, and the corresponding test report is provided.

All polypeptides were purified by reverse phase chromatography, and the molecular weight of the peptides was determined by mass spectrometry to determine whether the products were correct. The MS test results also showed most of the major impurities.

If necessary, net peptide content testing, such as amino acid analysis or elemental analysis, can also be provided.

These methods can confirm the amino acid composition of the polypeptide, and they can be used as complementary methods for polypeptide identification.

All peptides delivered have met the purity requirements of the customer.

Those that did not meet the purity requirements were discarded.

Of course, if the customer needs, you can also send it to them.


Q: How to explain the P+Na and P+K peaks in MALDI(MS)?

A: It is common to see Na and K peaks in MALDI. Sodium and potassium are derived from solvent water.

Even distilled and deionized water can contain trace amounts of sodium and potassium ions that cannot be completely removed.

They also ionize during mass spectrometry and bind to the free carboxyl groups of the peptides.

Because there is no system to purify water to remove sodium and potassium ions from the water, it is sometimes inevitable that there will be sodium and potassium peaks in the MALDI MS map.


Q: Why N-terminal acetylation and C-terminal amidation?

A: These modifications allow the peptide not to be degraded or to mimic the original state of its α-amino and carboxyl groups in the parent protein.


Q: How do I dissolve polypeptides in DMSO?

A: Dimethyl sulfoxide (DMSO) is a sulfur-containing organic compound with the molecular formula of (CH3)2SO. It is a colorless and odorless transparent liquid at room temperature.

DMSO is often used as a cryoprotectant in cell banks.

DMSO prevents intracellular/extracellular crystal formation during cell freezing and works at a concentration of 10%.

DMSO usually binds with salt or serum albumin.

Hydrophobic peptides can be easily dissolved in DMSO.

However, DMSO can increase the permeability of cells. If the polypeptide is dissolved in DMSO, it will have a toxic effect on cells.

High concentrations of DMSO should never be used in cell culture.

DMSO at a concentration of 5% dissolves the cell membrane.

Most cell lines can tolerate 0.5% DMSO and a few can tolerate 1% concentration without exhibiting severe cytotoxicity.

However, primary cell cultures are more sensitive to it.

So if the primary cells are used for the dose/response curve (feasibility), the concentration should be less than 0.1%.

For some very hydrophobic polypeptides, try dissolving them in a small amount of DMSO(30-50ul,100%) and slowly (drop by drop) adding them to a constantly stirred aqueous solution such as PBS or other desired buffer until the desired concentration.

If the peptide solution becomes cloudy during titration, the dissolution limit has been reached.

In addition, ultrasound helps to dissolve the peptides.

Experience:

DMSO at a concentration of 0.1% is safe for almost all cells.

DMSO, which is widely used in cell culture, has a final concentration of 0.5% and does not cause cytotoxicity.

Although 1% of DMSO is not cytotoxic in some cells, 0.5% is recommended.

In 5% of cases, DMSO has been used successfully in some cells.

The final concentration is always maintained at 0.5%, but it can be dissolved in 100% DMSO at a high concentration of 200 times when stored.


Q: Any recommendations for the design of phosphorylated modified peptides?

A: The efficiency of coupling from the phosphorylated amino acid decreases with increasing length.

The synthesis direction is from C to N, and it is recommended that no more than 10 residues remain after the phosphorylated amino acid, that is, no more than 10 residues remain before the number of amino acids phosphorylated from N to C.


Q: How to select N-terminal modification and C-terminal modification of polypeptide?

A: The default terminus of a peptide is an N-terminus free amino group and a C-terminus free carboxyl group.

The sequence of the peptide often represents the sequence of the parent protein. In order to be closer to the parent protein, the peptide terminal often needs to be closed, that is, N-terminal acetylation and C-terminal amidation. This modification avoids the introduction of excess charge, and also makes it more able to prevent the action of exonuclase, making the peptide more stable.


Q: I need a cyclic peptide that contains a tryptophan. Can it be oxidized?

A: Oxidation of tryptophan is a common phenomenon in the oxidation of peptides, and peptides are usually cycled and then purified. If the oxidation of tryptophan occurs, the retention time of the peptide on the HPLC column will change, so the oxidized peptide can be removed by purification.

In addition, oxidized peptides can also be detected by MS.


Q: How do you determine if the peptide has been cycled?

A: The Ellman reaction is generally used to test whether the ring reaction is complete.

If the Ellman test is positive (yellow), the reaction is incomplete;

If the test results are negative (not yellow), the ring reaction has been completed.


Q: Were the peptides containing Cys reduced prior to shipment?

A: If the peptide is not found to have been oxidized, we generally do not reduce Cys.

All peptides are obtained from crude products after purification and lyophilization at pH 2, which protects at least to some extent from oxidation of Cys.

Peptides containing Cys are generally purified at pH 2 unless there are specific reasons for purification at pH6.8.

If purification is performed at pH6.8, the purified product must be immediately treated with acid to prevent oxidation.

In the final quality control step, for the peptide containing Cys, if the molecular weight of (2P+H) is found on the MS map, it indicates that Cys has been oxidized to form a dimer.

If there is no problem with MS and HPLC, we will directly freeze dry and ship.

It should be noted that peptides containing Cys undergo slow oxidation over time, and the degree of oxidation depends mainly on the peptide sequence and storage conditions.


Q: Do we need a space between the peptide and the modified dye for fluorescent labeling?

A: most dyes are high molecular weight aromatic amino acids, in order to avoid the interaction between the peptide and the fluorescent label (for example, FITC easily binds to cysteine or lysine residues in the peptide sequence).

, to maintain the conformation and biological activity of the protein, we recommend the introduction of a flexible spacer, such as AHX, which is a ring structure containing 6 carbon molecules, to maintain the stability of the fluorescent label.

In general, dyes such as biotin and FITC can be labeled at either the amino terminus or the carboxyl terminus of the protein.

However, in order to synthesize peptides in the shortest possible time, with the most convenience and efficiency, Guto Bio recommends that customers select the label at the amino end.

Since the synthesis of a polypeptide usually starts at the carboxyl end, the amino end is then modified as the last step, requiring no special binding.

On the contrary, carboxyl end modification requires additional steps, so the process is more complex.


Q: What are the common salt forms of polypeptides?

In what way is desalination or desalination done?

A: Most peptides are isolated and purified under the TFA system, so the majority of peptides are in the form of TFA salts. Pharmaceutical peptides are usually in the form of acetate and hydrochloride. Few pharmaceutical peptides are in the form of special salts.

The methods of salt transfer are mainly ion exchange method and HPLC method, while desalination can be done by G25 (a kind of dextran gel from Ammatsia) column.

Acetate and hydrochloride prices are generally 20%-30% higher than TFA prices.


Q: What is the reason for the frequent baseline drift during peptide testing?

How to solve it?

A: Gradient elution with a fixed concentration of trifluoroacetic acid (TFA) can sometimes cause a shift in the absorption baseline at 210-220nm, which is responsible for many inverted phase separation baseline shifts.

To reduce or eliminate the baseline shift caused by the spectral absorption change of trifluoroacetic acid, the detection wavelength should be as close as possible to 215nm, and 15% less trifluoroacetic acid should be added in solvent B than in solvent A to compensate for the baseline shift.

For example, when the trifluoroacetic acid in solvent A is 0.1%, 0.085% can be used in solvent B.


Q: What are the factors that affect the results of the peptide purity test?

A: there are many factors that affect the results of polypeptide purity detection, mainly including mobile phase system, column type, column temperature, wavelength and chromatograph performance indexes, each of which may cause errors in the results.


Q: What mobile phase systems or ion pair reagents are available for the separation and purification of polypeptides?

A: At present, the most commonly used ion-pair reagent is trifluoroacetic acid (TFA). Trifluoroacetic acid regulates the pH of the eluent and also acts as an ion-pair reagent to interact with peptides, thus enhancing the separation effect and significantly improving the peak shape.

Other mobile phase systems or ion-pair reagents that can be used for separation and purification of polypeptides include acetic acid system, phosphoric acid system, hydrochloric acid system, sevafluorobutyric acid, etc., which can achieve good separation effect by adjusting pH appropriately.

Tel: +86-571-88211951 , 88211921. Fax:+86-571-88211907
Email: sales@gotopbio.com, Sales1@gotopbio.com
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