Application Notes - Delfia Protein Kinase Assays

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DELFIA®
Application Notes
DELFIA® Protein Kinase assays
4.Wash the plate six times with
wash solution using the DELFIA Platewash
(1296-024 or 026).
5. Add 200 µl of DELFIA Enhancement
Solution, shake for 10 min with Plateshaker
(1296-001/002 or 003/004) to guarantee a
stable signal level and measure the plate
with the Wallac time-resolved fluorometer.
zy
me
P
En
Protein kinases use ATP to phosphorylate
certain amino acid residues (tyrosine, serine
or threonine) on their substrates. In receptormediated signalling the phosphorylation
forms an important secondary messenger
system. Phosphorylation can be measured
following radioactive 32P incorporation,
which, however, involves the problems of
radioactive work, safety and disposal.
Eu-labelled anti-phosphotyrosine, -threonine
and threonine-proline are available for the
sensitive detection of phosphorylation levels
on various substrates using time-resolved
fluorometry and DELFIA® as the detection
technology.
Eu
Convenient collection of the substrates
(peptides, polypeptides or proteins) on
coated microtitration plates can be
accomplished using streptavidin plates for
biotinylated substrates or glutathione or
anti-glutathione-S-transferase plates for
substrates expressed with a GST tag.
B
P
Eu
PROTOCOL
B
Tyr
Antiphosphotyrosine
FIG.1. Detection of phosphorylation using
Eu-labelled anti-phosphotyrosine and streptavidincoated microtitration plates.
1. In the assay, first incubate the substrate
with enzyme (receptor), ATP and effectors.
2. After phosphorylation collect the
phosphorylated products on coated plates.
Incubate 1-2 hours and wash the plate two or
three times with wash solution.
3. Add Eu-(or biotin-) labelled antibodies in
DELFIA Assay Buffer (200 µl, 250 ng/ml) and
incubate for 1-2 h. If performed in the
indirect way, the secondary incubation with
Eu-streptavidin takes 20-30 minutes.
www.perkinelmer.com/lifesciences
ATP
The DELFIA plate
is coated with
poly-glutamyl-tyrosine
Streptavidin
Biotin
ADP
Eu3+
Tyrosine
kinase
FIG.2. Schematic assay for detection of phosphorylation using Eu-labelled streptavidin for quantitation of
biotinylated anti-phosphotyrosine antibody. For more information see Braunwalder et al.
1244-360
500
50
20
10
0.1
0.2
4002-0010
1244-114
2.0
1.5
1244-105
1.0
4001-0010
0.5
0.0
1
10
100
Phosphopeptide 1 (pmol/ml)
3.5
1
2
5
Phosphopeptide ( pmol/ml )
1000
10
FIG.3. Quantitation of phosphorylated phosphopeptide using Eu-labelled anti-phosphotyrosine.
WALLAC PRODUCTS
In addition to the DELFIA system the following
specific reagents are available:
AD0038
Eu-labelled anti-phosphotyrosine,
(PY20), 50 µg (for ten plates)
AD0040
Eu-labelled anti-phosphotyrosine,
(PT66), 50 µg (for ten plates)
AD0092
Eu-labelled anti-phosphothreonine,
10 µg (for 1-2 plates)
AD0097
Eu-labelled anti phosphothreonineproline, 10 µg (for 1-2 plates)
AAAND-0005 Streptavidin-coated
yellow plate, 10 plates
CC11-B10
Streptavidin-coated white
384 plate, 10 plates
CC41-1210
Anti-GST coated yellow plate,
10 plates
20
1420
1296-026
1296-003/004
1296-014
1296-016
1296-017
VICTOR2™ Multilabel counter
DELFIA Platewash
DELFIA Plateshake
Multipette
Combitips, 5 ml, pack of 100
Combitips, 2.5 ml, pack of 100
PERKINELMER LIFE SCIENCES
ASSAY SERVICE
Eu-labelled anti-phosphoserine antibody service
REFERENCES
Assays are currently routinely used in a number of
drug discovery laboratories in the UK and USA.
Braunwalder et al. Measurement of the protein
tyrosine kinase activity of c-src using
time-resolved fluorometry of europium chelates.
Anal. Biochem. 238, 159-164 (1996).
Visit lifesciences.perkinelmer.com/referencedb
for more references.
For more information please contact your local
sales representative.
World Headquarters: PerkinElmer Life Sciences, 549 Albany Street, Boston, MA 02118-2512 USA (800) 551-2121
European Headquarters: PerkinElmer Life Sciences, Imperiastraat 8, B-1930 Zaventem, Belgium, +32 2 717 7911
DELFIA is a registered trademark of PerkinElmer, Inc.
www.perkinelmer.com/lifesciences
Printed in Finland by Offset House Oy Naantali 2001
100
1234-968-03, May 2001
300
Absorbance (405/490 nm)
( cps x 1000 )
1244-111
Eu-labeled streptavidin,
(for 50-125 plates)
DELFIA Assay Buffer, 250 ml
(for 5 plates)
DELFIA Assay Buffer, 1000 ml
DELFIA Wash concentrate,
25 x concentrate, 250 ml
DELFIA Enhancement
Solution, 250 ml
DELFIA Enhancement
Solution, 1000 ml
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