Biomedical Engineering Reference
In-Depth Information
poral behaviour of signalling pathways by subjecting labelled cells
to a time course of activation. This strategy has been used to pro-
file the time course of phosphorylation changes following epi-
dermal growth stimulation, either by specifically targeting phos-
When investigating protein-protein interactions SILAC has also
emerged as a useful tool. As discussed further in
Chapter 16
of this volume, for this purpose SILAC is used to discriminate
between specific biological interactions and unspecific binding,
an application that was initially demonstrated for the activated
identify proteins interacting specifically with Histone H3 depend-
stringent limitation of SILAC is that its application to tissue sam-
ples is not as straightforward as for its use to cells grown in culture
however, significant progress in this field has recently been made
labelling of proteins with SILAC reagents and for their analysis by
LC-MS/MS.
2. Materials
2.1.CellCulture
1. For all buffers and solvents listed in this chapter use Milli-Q
water, unless specified otherwise.
2. Dulbecco's Modified Eagle Medium (DMEM) deficient in
lysine and arginine (Invitrogen, Carlsbad, CA).
3. Dialysed Fetal Bovine Serum (FBS) (Gibco-Invitrogen,
Carlsbad, CA).
4. Normal “light” amino acids: L-lysine
(Lys0)
and
L-arginine
(Arg0) hydrochloride
(Sigma Chemicals,
Copenhagen, Denmark).
5. Stable isotope-labeled “heavy” amino acids: L-arginine-
13
C
6
hydrochloride (Arg6) (Cambridge Isotope Labs,
Andover, MA), L-arginine-
13
C
6
,
15
N
4
hydrochloride
(Arg10) (Sigma-Isotec, St. Louis, MO), L-lysine-4,4,5,5-
d
4
hydrochloride (Lys4) (Sigma-Isotec; cat. no. 616192),
and L-lysine-
13
C
6
,
15
N
2
hydrochloride (Lys8) (Sigma-
Isotec).
6. L-Glutamine (200 mM stock solution), penicillin/
streptomycin (10,000 U/10,000
g stock solution), and
trypsin-EDTA solution (trypsin, 200 mg/L and Versene-
EDTA 500 mg/L) (all from Gibco-Invitrogen).
μ
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