Biomedical Engineering Reference
In-Depth Information
activity, i.e. enzyme units (
mol of phospho-Aktide produced per
unit of time in minutes) per milligram of protein in the studied
samples.
μ
4. Notes
1. This assay can be performed with any cell line to be studied.
Here we exemplify the approach by using the acute myeloid
leukaemia cell line P31/FUJ.
2. Routine solutions should be prepared in water that has
a resistivity of 18 M
-cm, unless otherwise stated. The
resuspension solution used straight before running the
samples in the LC-MS along with all the solvents used in
the LC-MS system must be LC-MS grade. LC-MS grade
solvents used here were provided by LGC Promochem
(Middlesex, UK), but any other LC-MS grade solvents
would be adequate.
3. Ideally one should test the specificity of the enzymatic
assay; for this purpose a compound that inhibits directly
or indirectly the kinase to be tested can be used. In our
case, the use of Wortmannin, a specific covalent pan-
inhibitor of phosphoinositide 3-kinases (class I, II and III
reduces the amount of phospho-Aktide produced in the
reaction. Wortmannin is a very potent PI3K inhibitor, even
more so than LY294002, another commonly used PI3K
pan-inhibitor (IC50 s 5 nM and 1.4
μ
M, respectively)
4. The lysis buffer used in this protocol is Triton X-100 based.
Triton X-100 is a non-ionic detergent that intercalates in
the lipid bilayers disrupting the cellular structure and act-
ing as a lysing agent. This compound is very viscous at
room temperature; hence it is easier to use after being gen-
tly warmed.
5. For this kind of experiment, in which we study protein
kinase activities, it is essential that the lysis buffer con-
tains different protease and phosphatase inhibitors such
as Na
3
VO
4
and NaF (tyrosine phosphatase inhibitors);
aprotinin, PMSF and TLCK (serine protease inhibitors);
leupeptin (thiol and serine proteases inhibitor); pep-
statin A (acid proteases inhibitor) and okadaic acid (ser-
ine/threonine phosphatase inhibitor).
6. Any other reagent for protein quantification would be valid
as well.
7. The peptide used in the assay RPRAATF peptide is a
highly selective substrate of protein kinases downstream
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