| | Re: Kinase substrate Prediction
Protein Kinase A immuno-cathalytic determination
Phosphorylation and dephosphorylation of proteins, which are catalyzed by protein kinases and protein phosphatases respectively, are reported to regulate major cell functions. Therefore the measurement of kinase activities is indispensable for study of cell function. For the measurement PKA activity an indirect method we suggest in which an biotinylated tau unphosphorylated peptide (tau207-219) biotin-Ttds-GSRSRTPSLPTPP-NH2 is phosphorylated in serine residue, in presence of ATP, from PKA present in the sample. The amount of the peptide phosphorilation is directly correlated to PKA amount in the sample. The biotinylated phoshorilated peptide is captured by specific antibody, against phosphorylated form, immobilized on the wells MTP surface and the complex identified through complex streptavidin-HRP and determined by TMB substrate.
Coated plate : dispense 100 µl of 100-200 ng/well rabbit antibody anti-TAU(pT212) [Invitrogen cod. 44-740G] in PBS and incubate at 4 °C for 16 h. Drain the wells and dispense 200 µl blocking solution (2% BSA fraction V, modified Cohn, in PBS [140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4]) and incubate for 1 h at room temperature. Wash once with PBS, dry and store at 4 °C in bag with essiccant.
Biotinylated substrate peptide (BSP) : synthetic biotinylated peptide GSRSRTPSLPTP solution (Biotin-Ttds-GSRSRTPSLPTPP-NH2 (JPT Peptide Technologies - Berlin Germany) diluted in PBS (see above).
Reaction buffer : Hepes buffer 25 mM at pH 7.0, contained 5 mM MgCl2 and 0.5 mM di dithiothreitol
ATP (0.1 M) : dissolve 60 mg ATP in 0.8 ml H2O. Adjust the pH to 7.0 with 0.1 M NaOH. Adjust the volume to 1 ml with H2O. Dispense the solution into small aliquots and store at -20 °C. The molar absorption coefficient of ATP ε 259 = 15.4x103 at pH 7.0. Just prior to in assay use, dilute 0.1 M ATP with dH2O for obtain 100 µM ATP solution.
Stoping A : 10 mM EDTA
Wash solution : 0.1% Tween-20 in PBS 0.01 M at pH 7.4
Streptavidin-HRP (SA-HRP) molar ratio (5:1) : dissolved in NaHCO3 buffer 0.05M at pH 8.3, contained 0.05% thimerosal must be diluted just before use in PBS (see above).
TMB one bottle : dissolve in 950 ml dH2O, 3.6 g citric acid monohydrated, 1.838 g trisodium citrate dehydrate, when all dissolved add 1.442 g bacitracin and then 0.564 g urea hydrogen peroxide in continuous stirrer for all to dissolve. In other beaker, dissolve completely 300 mg 3,3',5,5'-Tetramethylbenzidine in 50 ml of dimethyl sulfoxide before adding to the above solution. After thorough mixing in an amber bottle the solution check pH, that must be about 3.4, and store at 4 C, stable 1 year.
Stoping B : 1 M H3PO4
Enzymatic reaction: to set in test-tube 10 µl of serum 100 µl of reaction buffer, 40 µl BSP diluted and 50 µl of ATP 100 µM solution and incubate at 30 °C for 30 min. Stop reaction dispensing 10 µl of stoping A solution. Mix and to store the test-tube at -20 °C up to moment of the immunometric determination.
Immunometric reaction : thaw contents tube at room temperature and then dispense 100 µl in coated plate and to incubate for 2 h at room temperature. Wash twice or more wells with washing solution and to distribute 100 µl of a solution 25 ng/ml of streptavidin-HRP and incubate for 2 h at room temperature. Wash as above and to distribute 100 µl of TMB one bottle and incubate for 15 mins. When the allotted time expires, to stop reaction with 100 µl of stoping B and then read the Abs to 450 nm (reference to 620 nm). Calculate against calibration curve using PKA standards solution (Sigma cod. P2645).