Application
The typical use for this product is in removing adherent
cells from a culture surface. The concentration of trypsin necessary to
dislodge cells from their substrate is dependent primarily on the cell type and
the age of the culture.
Trypsin-EDTA solution is used for the following applications:
- Used
as a supplement in cell culture for their maintenance[1]
- In
harvesting cells grown to confluence[2]
- to
detach lentivirus-transduced macrophages[3]
Biochem/physiol Actions
Trypsin cleaves peptides on the C-terminal side of lysine
and arginine residues. The rate of hydrolysis of this reaction is slowed if an
acidic residue is on either side of the cleavage site and hydrolysis is stopped
if a proline residue is on the carboxyl side of the cleavage site. The optimal
pH for trypsin activity is 7-9. Trypsin can also act to cleave ester and amide
linkages of synthetic derivatives of amino acids. EDTA is added to trypsin
solutions as a chelating agent that neutralizes calcium and magnesium ions that
obscure the peptide bonds on which trypsin acts. Removing these ions increases
the enzymatic activity.
Serine protease inhibitors, including DFP, TLCK, APMSF, AEBSEF, and aprotinin,
amongst others, will inhibit Trypsin.
Preparation Note
Incubating cells with too high a trypsin concentration for a
long period can damage cell membranes and kill the cells. Solubilizing trypsin
or diluting it from a concentrated solution should be done with a buffered salt
solution contaiing no Ca2+ or Mg2+.
Other Notes
Trypsin consists of a single chain polypeptide of 223 amino
acid residues, produced by the removal of the N-terminal hexapeptide from
trypsinogen which is cleaved at the Lys - lle peptide bond. The sequence of
amino acids is cross-linked by 6 disulfide bridges. This is the native form of
trypsin, beta-trypsin. BETA-trypsin can be autolyzed, cleaving at the Lys - Ser
residue, to produce alpha-trypsin. Trypsin is a member of the serine protease
family.
Disclaimer
This product is stored frozen between -10 and -40°C.
Repeated cycles of freezing and thawing should be avoided.
