Drug Discovery | Research | Clinical Screening 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