Assay Method Information |
|
| Enzymatic Assay |
Description: | A peptide mobility shift assay was used to quantify the phosphorylation of the JAKtide (JAK2 and JAK3) or the IRS-1 peptide (JAK1 and Tyk2). Reactions were carried out in a 384-well plate (Matrical MP-101) in a 10 L total volume. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween-20, ATP (4 M for JAK2 and JAK3, 40 M for JAK1 and 7 M for Tyk2)), 2% DMSO and 1 M peptide substrate (JAKtide for JAK2 and JAK3 and IRS-1 peptide for JAK1 and Tyk2). Compounds were diluted serially in 100% dimethyl sulfoxide and tested in an 11 point dose response in duplicate or quadruplicate (200 nl of compound/DMSO was added per 10 L reaction). The reactions were initiated by the addition of enzyme to the final concentration of 2 nM JAK2, 1 nM JAK3, 12 nM Tyk2 or 20 nM JAK1. The assay was run for 240 minutes for JAK1, 150 minutes for JAK2, 90 minutes for JAK3 and 70 minutes for Tyk2. |
Affinity data for this assay | |
---|---|
If you find an error in this entry please send us an E-mail |
Home |
| |
Search |
| |
Deposit |
| |
SiteMap |
| |
About us |
| |
Email us |
| |
Info |
|
©2000 BindingDB. All rights reserved. |