On 9 May 1995, Anne Spang wrote:
> Hi!
> I=B4m looking for a protocol for endoproteinase lys-C digest!
> Any help will be appreciated!!
>=20
> Thanx
> Anne
>=20
>=20
Hi,
=09Endoproteinase Lys-C should work in any buffer at a pH between 7.5
and 9.5. However, 100mM amm. bicarbonate, pH9 is traditionally used with
an enzyme to subs. ratio of 1:100 (w/w) and at a temp. of 37C. Digestion
is also facilitated with the addition of SDS to a final concentration of
0.01%. Since the preparation of the protein to be digested is also a
factor, timed aliquots of the digestion mix may be checked on SDS-PAGE.=20
Certain compounds eg. DFP and TLCK will inhibit the enzyme. However, the
enzyme works fine in the presence of EDTA and PMSF at the concentrations
used for normal protein work (ie. 1 - 2 mM).=20
Hope this helps,
with regards,
HSR.
=09=09=09>>>>>>>>>>>>>>>>>>>>>>>>>>>
=09=09=09 Hiranya S. Roychowdhury
=09=09=09 Plant Genetic Engineering Lab.
=09=09=09 Box 3GL, NM State Univ.
=09=09=09 Las Cruces, NM 88003
=09=09=09 Phone: (505) 646-5785
=09=09=09 hroychow at nmsu.edu
=09=09=09<<<<<<<<<<<<<<<<<<<<<<<<<<<
