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PURIFICATION
OF SYNTHETIC PEPTIDES Most
of the synthetic peptides prepared by the LMAP are free of other peptide
contaminants. This is documented by
the mass spectral analysis and the amino acid analysis. The peptides can usually be used as is for antigen
preparation. However, you may wish
to clean up additional peptide for other experiments, such as for use in tissue
culture. The simplest purification
procedure is a simple desalting column. This
will remove the small molecule scavengers used to protect the amino acid side
chains during cleavage of the peptide from its resin.
If you wish to HPLC purify your peptide for other experimental purposes
and do not have an instrument in your laboratory, we can train you to use the
LMAP preparative Rainin HPLC. LMAP
staff can also do this for you. If
you have any problems with peptide solubility, desalting, or HPLC separations,
we will be glad to answer your questions. Desalting
of Synthetic Peptides:
Desalting of peptides is performed on short columns of Sephadex G-10/G-25
or BioGel P-4/P-10 in a volatile solvent which can later be removed by
lyophilization or SpeedVac concentration. Most
soluble peptides can be desalted in 20% acetic acid.
If you are not certain of the solubility of your peptide, place small
amounts of the peptide in several microcentrifuge tubes.
Try to dissolve the peptide in several of the following solvents:
5% acetic acid, 20% acetic acid, 5% formic acid, 25 mM ammonium
bicarbonate. Choose the most
effective solvent for your peptide. A
large amount of peptide can be desalted on a column of Sephadex G-10 (2.5 x 15
cm). Wash the column with 5 bed
volumes of the selected solvent. Dissolve
up to 250 mg of peptide in 10 ml of solvent.
Load the peptide gently onto the surface of column.
After absorption, add 3 ml of solvent, gently washing the walls without
disturbing the column bed. Then
elute with the solvent, collecting 2 ml fractions.
The peptide should elute at the void volume of the column.
Monitor the absorbance at 220 nm (for peptide bonds) or 280 nm (for Trp
or Tyr). Alternatively, use one of
the peptide quantitation assays described in the next section to locate your
peptide. If
you do not have experience with packing gel filtration columns, prepacked
columns are available from BioRad Laboratories or Pierce Chemical Co.
These columns will be shorter, but the sample may be as much as 1/3 of
the column volume. Resolution is
best if the sample is no more than 20% of the column volume.
HPLC
Purification of Synthetic Peptides:
A simple linear gradient from 0.1% trifluoracetic acid (TFA, Pierce,
Sequenal Grade) to 70% acetonitrile (+ 0.1% TFA) will purify most peptides on
reversed phase columns. A very
soluble peptide can be separated on a C-18 column.
However, longer or more hydrophobic peptides are more effectively eluted
from C-4 or C-8 columns. Many
peptides are not soluble in dilute TFA solution, making them difficult to
prepare for injection onto an HPLC column.
Instead, peptides can be dissolved in 6 M guanidine hydrochloride
(Pierce, Sequenal grade) containing 0.1% TFA [1g guanidine + 1 ml water yields a
6 M solution]. The guanidine salts
will elute in the breakthrough of the column, while the peptide will bind.
Monitor the column effluent at 220 nm.
The major peak will be your peptide.
There will be minor ultraviolet absorbing peaks, which are the scavenger
reagents. Please
note that cysteine-containing peptides may sometimes give split peaks,
representing both oxidized and reduced forms of the peptide.
However, because of the large amounts of reducing agents and scavengers
present during cleavage from the resin, most peptides are in the fully reduced
state. Sep-Pak
Cartridges for Peptide Desalting (Solid Phase Extraction):
Small,
disposable cartridges containing reversed phase resin are available from several
manufacturers. These operate on the
same principles as HPLC-type separations, but do not involve pumping systems,
just disposable syringes. Instructions
are available with these inexpensive packages.
The peptides are eluted in either methanol or acetonitrile containing a
small amount of trifluoracetic acid. These
solutions can be used as stocks, or can be easily dried down in a vacuum
concentrator. Extra drying time is needed to remove trifluoracetic acid.
FPLC Columns: Many laboratories at AECOM have FPLC systems. There are several prepacked FPLC columns which are useful for peptide cleanup. The PepSep column is a reversed phase column run under conditions similar to those described above for HPLC reversed phase. The Fast Desalting column is a gel filtration column which can desalt your peptide in 10-15 minutes. Although these FPLC columns are somewhat expensive, Pharmacia now sells a set of 5 HiTrap Desalting columns. These inexpensive columns can be run with either a syringe, a peristaltic pump, or an FPLC pump. They can even be used in series, if the sample to be desalted exceeds the capacity of a single column. |