long do peptide last in the fridge? How should peptides be stored?
For long-term storage the peptide should be kept in solid form in
the deep freezer at < -15 °C. If stored at room temperature some
peptides containing methionine or cysteine may begin to degrade. Therefore, we
recommend storing them at -20C as soon as possible after receiving the package.
At -20 or -80, the peptides will remain potent for 6 months or years before
beginning to degrade. For short-time storage a refrigerator (+4 °C) will
suffice. Peptides should be protected from intense sunlight. Peptides
containing fluorophores should be kept in the dark.
may happen during the peptide storage?
Some typical degradation reactions or
racemization may include the oxidation of Met, Trp, Tyr, or Cys.
The deamidation of Asn, Gln, and the C-terminal amide may happen. The
aspartimide may form. There might be the cleavage of Asn-Pro. The N-terminal
Gln may form pyroglutamine. The dimerization of Trp and Tyr may form.
peptides soluble in water?
is a list of non-polar, hydrophobic,
or water-fearing amino acids: Ala (A), Ile (I), Leu (L), Met (M), Phe
(F), Pro (P), Trp (W), Val (V).
Peptides containing 50% and more
hydrophobic or nonpolar residues as above might be insoluble or only
partly soluble in aqueous solutions. In this
case, dissolve the hydrophobic peptides in the small volume
of 50% (v/v) DMSO, DMF or acetonitrile in water first. Then add water or
buffer to the desired concentration.
is the best way to keep synthetic peptide in solution?
Dissolved peptides are less stable than the lyophilized dry
powder. The solutions should be aliquoted before freezing to avoid thawing-refreezing
cycles. Peptides containing Asn, Gln, Cys, Met, Trp, Tyr may be less stable
because of the possible oxidization. The stock solutions should be in dry
organic solvents to avoid premature hydrolysis. The buffer of pH 5-7 is
considered as optimum for the stability of the aliquots. The peptide (>95%
HPLC purity) solutions with cell cultures are frequently used without any
sterilization. If the experiments are typically as short as a few
hours, the bacterial contamination wouldn’t be a problem.
Why do the dissolved peptides lose their activities? How to prevent peptides from losing their bioactivities?
The oxidation of Methionine yielding the sulfoxide might be the main reason for losing the peptide activity. The rate of sulfoxide formation is sequence-dependent. The best way is to replace the Methionine with its stable isostere Nle. Sulfotyrosine-containing peptides may lose their activity due to the desulfation.
peptides containing free Cys supplied as monomers? Will the Cys-containing Peptides be easily
LifeTein provides Cys containing peptides as
monomers unless specified otherwise. As air oxidation cannot be completely
prevented, reducing the peptide before use by treatment with dithiothreitol
(DTT) may be helpful to your experiment.
to dissolve peptides containing disulfide bridges? How do I know if a cysteine in a peptide is
free or oxidized?
Basic buffers should be avoided for peptides
containing disulfide bridges.
Peptides containing free thiol group may oxidize to form dimers or oligomers during
storage, even as the lyophilized dry powder at a low temperature. Peptides
provided as acetate salt are more sensitive to Cysteine oxidation than the
corresponding TFA salt or HCL salt. The disulfide bond formation is rapid at
neutral or slightly basic pH. Disulfide bridge formation is reversible. The
disulfide bonds can be reduced at basic conditions using DTT. The pH 7-9.5 is
the optimum pH-range for reductions with DTT. The DTT solutions should be
freshly prepared because DTT is readily oxidized.
to dissolve peptides containing free cysteines?
Peptides containing a single free cysteine will be oxidized at
pH>7 to form dimers. This oxidation can be reverted. Peptides containing two
or more thiol moieties may yield a mixture of products upon oxidation. The pH
7.5-8 is the best condition for the disulfide bond formation. Hence, peptides
containing free cysteines are best dissolved in degassed solvents, e.g. buffers
pH<7, diluted acetic acid, 0.1% trifluoroacetic acid in aqueous
acetonitrile. DMSO should be avoided, especially with peptide
How to dissolve amyloid beta amyloid peptide (1-42)?
The amyloid peptide Aβ (1-42), or other amyloid mutants
may form insoluble aggregates during the storage. Aβ (1-42) is soluble in
hexafluoroisopropanol (HFIP), DMSO, 0.1% aqueous ammonia, 50 mM TRIS ∙ HCl, or
1mM NaOH. Reconstitution in HFIP or DMSO takes time whereas ammonia rapidly
dissolves the peptide. The volatile solvent HFIP is usually evaporated leaving
a residue of monomeric, soluble Aβ (1-42), which can be reconstituted with the
chosen buffer at pH 7.4 to induce fibrillation. Aβ
(1-42) solutions in DMSO or aqueous bases can be diluted directly with working
How to dissolve amyloid beta protein (1-40)?
The freshly made Aβ (25-35) and (1-40) are
soluble in oxygen-free water. Some old peptides may require the addition of
acetic acid for dissolution. Do not dissolve the
amyloid peptides in PBS. It is better to try water or 50% acetic acid and then
dilute with the working buffer.
dry ice required for the shipment of peptides?
Dry ice is not required for the shipment of
peptides. The lyophilizates in vacuum sealed vials are sufficiently stable to
tolerate 3-5 days at ambient temperature. Upon receipt, the product should be
stored at the recommended storage temperature of -20 or -80.
I convert a free peptide or peptide with free acid into the peptide amide?
Unfortunately, the C-terminal carboxylic group cannot
be converted to a carboxamide once the peptide is synthesized. The peptide
amide has to be synthesized and cleaved in a different method.
expect batch-to-batch variability?
The impurity profile and especially the peptide
content can vary from batch to batch, even if we use the same the standard protocols
of synthesis and purification.
is the difference between research-grade and GMP-API?
The Research-grade peptides are for laboratory
and research purposes only!
They may not be used as drugs for humans. The GMP-APIs
are peptide products applicable to humans.
are peptides sold as salts?
Most peptides contain the basic functionalities: the guanidino
group of Arg, the ε-amino group of Lys, the free N-terminus, and the imidazole
moiety of His. These basic functionalities can form salts with acids. All
our peptides are provided as trifluoroacetate salts unless specified otherwise.
During cleavage from the carrier resin
and purification, the peptide will react to the trifluoroacetic acid (a strong
The additional ion-exchange steps are needed to make the acetate
salt or HCL salt form peptides. Some acidic
peptides, containing Asp, Glu, phosphor group or sulfotyrosine, can form salts
with bases and may be provided as the ammonium salts.
do you purify the peptides? How pure are your peptides?
Peptides obtained by SPPS are usually purified by
the preparative RP-HPLC. The HPLC-purity of most of our peptides is 95% or
above. The lower purities may be expected for peptides containing free Cys, an
N-terminal Gln, or sulfated peptides. The purity could be improved with the
optimized synthetic and purification methods.
do you synthesize small peptides?
Dipeptides, tripeptides and other short peptides
are usually synthesized in the solution called Liquid Phase Synthesis.
do you synthesize peptides?
Fmoc- SPPS (solid-phase peptide synthesis) is the main method. The peptide is elongated starting from the C-terminus to
the N-terminus of the sequence. An inert,
insoluble but swellable polymer resin is used and the amino acids are added in
the C→ N direction. After each coupling step, the Nα-blocking group is removed through
repetitive protection and deprotection steps. LifeTein’s peptide synthesis
method is fully automated.
do you denote amino acids?
H- stands for an N-terminal free amino moiety,
-OH for the unmodified C-terminal carboxyl group. H-Hyp-OH
stands for – L-cis-hydroxyproline; H-Nle-OH
is – L-norleucine;
Pyroglutamyl is abbreviated as Pyr. The natural peptides consist of L-amino acids. The D-enantiomer and the
racemate are designated by -D- and -DL-, respectively.
What do L- and D-enantiomer mean?
All α-amino acids are asymmetric molecules except glycine. They
can be obtained in two forms called enantiomers. Enantiomers differ
considerably in their biological activity.
The D-enantiomer (D: dexter) represents the mirror image of the
L-enantiomer (L: laevus). D- and L-enantiomer do not differ in their physical
properties. Most L-amino acids rotate the light counterclockwise.
to dissolve the peptide?
of Amino Acids:
Arg (R), His (H), Lys (K)
(hydrophobic): Ala (A), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Trp (W),
uncharged: Asn (N), Cys (C), Gly (G), Gln (Q), Ser (S), Thr (T), Tyr (Y)
Acidic: Asp (D), Glu (E)
solubility of a peptide is primarily dependent on the physical properties of
its amino acids.
number of basic amino acids including the N-terminal amino group is more than
the number of acidic amino acids including the C-terminal carboxyl moiety. The
basic peptides may be dissolved in a small amount of an acidic solvent such as
acetic acid or trifluoroacetic acid (TFA) and then diluted to the desired
number of acidic amino acids including the C-terminal carboxyl group is more
than the number of basic amino acids including the N-terminal amino group. The acidic peptide may be reconstituted in a
small amount of a basic solvent such as 0.1% aqueous NH3 and then diluted with
water to the desired concentration. The peptides containing free cysteines
should be dissolved in the degassed acidic buffers because the thiol moieties could
be rapidly oxidized at pH > 7 to disulfide bonds.
Neutral or highly
hydrophobic peptides containing a high proportion of polar uncharged amino
acids and/or hydrophobic amino acids should be dissolved in a small amount of
an organic solvent such as DMSO, DMF, acetic acid, acetonitrile, methanol,
propanol, or isopropanol and then diluted with water (or buffer) to the desired
agents, such as urea or guanidinium hydrochloride may be used to solubilize
peptides which tend to aggregate. The reconstitution of the aggregated peptide
may take up to several hours. Sonication for several minutes in a water bath
may be helpful to accelerate the dissolution of larger particles. The peptides
containing Trp, Met or Cys residues require special care to avoid oxidation.
is peptide folding? Why do polypeptide chains fold?
Peptide folding is the process by which
a peptide structure assumes its functional shape or conformation. By
coiling and folding into a specific three-dimensional shape, the
peptides are able to perform their biological function. The amino acids with
hydrophobic side chains tend to end up clustered at the core of the structure
so that they are out of contact with water. Covalent disulfide
bridges can affect the shape of a protein. Protein
folding is aspontaneous process because the Gibbs free energy is
are the four levels of protein folding?
There are four stages of peptide or protein
folding, primary, secondary, tertiary and quaternary. The secondary structure
is the protein beginning to fold up. It can have two types
of structure: the alpha helix, a coil shape held by hydrogen bonds in the same
direction as the coil.
does peptide unfold at high temperatures?
Heat can be used to disrupt hydrogen bonds and
non-polar hydrophobic interactions. This occurs because heat increases the
kinetic energy and causes the molecules to vibrate so rapidly and violently
that the bonds are disrupted.
factors can disrupt the peptide structure and
thus denature the peptide or protein?
Changing in pH and salt concentration will alter electrostatic
interactions between charged amino acids. Higher temperatures reduce the
strength of hydrogen bonds.
The presence of the reducing agents will break disulfide bonds
However, none of these agents breaks peptide bonds, so the
primary structure of a protein remains intact when it is denatured.