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:6/22/2017)... Colorado (PRWEB) , ... June 21, 2017 , ... ... RTP regional office in North Carolina, and engages Timothy Reinhardt to manage the ... of quality leadership at Pfizer Inc, with his most recent role as the ...
(Date:6/22/2017)... ... June 22, 2017 , ... AESKU.GROUP, ... DST Diagnostische Systeme & Technologien GmbH, thereby expanding its product portfolio to include ... hay fever, urticaria, asthma, atopic eczema or a food allergy. Allergies are escalating ...
(Date:6/22/2017)... ... June 22, 2017 , ... For the months of May ... Spotlight series on “Cell Therapy Regulation” for its regenerative medicine followship. ... the unique regulatory challenges of stem cell medical research. , Stem cell clinical ...
(Date:6/22/2017)... Yorba Linda, Ca (PRWEB) , ... June 22, 2017 , ... ... labs worldwide. It took 20 years until the first data on cross-contamination of human ... cell lines has been an increasing issue in cell culture labs and is associated ...
Breaking Biology Technology:
(Date:5/16/2017)... -- Veratad Technologies, LLC ( www.veratad.com ), an innovative and ... solutions, announced today they will participate as a sponsor ... May 17, 2017, in Washington D.C.,s ... Identity impacts the lives of billions of ... digital world, defining identity is critical to nearly every ...
(Date:4/19/2017)... 19, 2017 The global military ... is marked by the presence of several large global ... by five major players - 3M Cogent, NEC Corporation, ... for nearly 61% of the global military biometric market ... the global military biometrics market boast global presence, which ...
(Date:4/13/2017)... April 13, 2017 According to a new market ... Identity Analytics, Identity Administration, and Authorization), Service, Authentication Type, Deployment Mode, Vertical, ... Market is expected to grow from USD 14.30 Billion in 2017 to ... of 17.3%. ... MarketsandMarkets Logo ...
Breaking Biology News(10 mins):