Navigation Links
DELFIA Protein Kinase assays.

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.

PROTOCOL

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.

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.

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.

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.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. A DELFIA Assay for Inhibition of PARP
2. A New C-Terminal GST Vector for Protein Production in S. pombe
3. New Mammalian Two-Hybrid System Detects Protein-Protein Interactions
4. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
5. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
6. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
7. Optimized Imaging of Protein Gels Stained with Coomassie Brilliant Blue Dye
8. High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
9. Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells
10. Yeast Protein Production System Features High Yields and One-Step Purification
11. Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:4/29/2016)... ... April 29, 2016 , ... Amendia, Inc., a ... procedures, today announced the completion of a significant transaction and partnership that positions ... customers and partners. Kohlberg & Company, L.L.C. (“Kohlberg”), a leading private equity ...
(Date:4/29/2016)... 2016 According to a ... "Separation Systems for Commercial Biotechnology Market - Global ... 2015 - 2023", the separation systems for commercial ... in 2014 and is projected to expand at ... to reach US$ 19,227.8 Mn in 2023. ...
(Date:4/29/2016)... ... April 29, 2016 , ... ... to transform technology into a viable company, CereScan’s CEO, John Kelley, joined other ... a recognized leader and mentor in the Denver area business community, shared his ...
(Date:4/29/2016)... Louisville, KY and San Diego, CA (PRWEB) , ... April 29, ... ... the National Stem Cell Foundation (NSCF) to support the development of a patient-specific stem ... by Dr. Andrés Bratt-Leal in the lab of Dr. Jeanne Loring at The Scripps ...
Breaking Biology Technology:
(Date:4/28/2016)... SAN FRANCISCO and BANGALORE, India ... part of EdgeVerve Systems, a product subsidiary of Infosys (NYSE: ... service provider, today announced a global partnership that ... convenient way to use mobile banking and payment services. ... Mobility is a key innovation area for financial services, but ...
(Date:4/28/2016)... First quarter 2016:   , Revenues amounted ... quarter of 2015 The gross margin was 49% (27) ... the operating margin was 40% (-13) Earnings per share ... operations was SEK 249.9 M (21.2) , Outlook   ... M. The operating margin for 2016 is estimated to ...
(Date:4/26/2016)... BANGALORE, India and LONDON ... Infosys Finacle, part of EdgeVerve Systems, a ... ), and Onegini today announced a partnership to ... banking solutions.      (Logo: http://photos.prnewswire.com/prnh/20151104/283829LOGO ... banks to provide their customers enhanced security to ...
Breaking Biology News(10 mins):