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BioAssay: AID 624057

Human p-Tau and Total Tau ELISA Measured in Cell-Based System Using Plate Reader - 2133-07_Inhibitor_Dose_DryPowder_Activity

The glycogen synthase kinase-3 beta (GSK-3b) is a known master regulator for several cellular pathways and plays a critical role in metabolism, transcription, development, cell survival, and neuronal functions. The overall objective is to identify one or multiple series of binders of GSK-3beta with sub-micromolar potency. Such compounds will become probe(s) with demonstrated kinase-selectivity. more ..
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 Tested Compounds
 Tested Compounds
All(21)
 
 
Active(17)
 
 
Inactive(5)
 
 
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 Tested Substances
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Active(19)
 
 
Inactive(5)
 
 
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 Related BioAssays
AID: 624057
Data Source: Broad Institute (2133-07_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2012-04-10
Hold-until Date: 2013-04-07
Modify Date: 2013-04-10

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 17
Related Experiments
Show more
AIDNameTypeComment
2119Summary of Probe Development Efforts to Identify Inhibitors of Glycogen Synthase Kinase-3 beta (GSK-3b)Summarydepositor-specified cross reference: summary assay
2097Cell-Free Homogeneous Primary HTS to Identify Inhibitors of GSK3beta ActivityScreeningsame project related to Summary assay
434947Luminescence Cell-Free Homogeneous Counter Screen to Identify Inhibitors of ADP-glo ReagentsConfirmatorysame project related to Summary assay
434954Luminescence Cell-Free Homogeneous Dose Retest to Identify Inhibitors of Glycogen Synthase Kinase-3 beta ActivityConfirmatorysame project related to Summary assay
435010Fluorescence Cell-Free Homogeneous Dose Retest to Identify Inhibitors of RecA-Intein Splicing ActivityConfirmatorysame project related to Summary assay
623978Inhibition of Human GSK3 alpha Activity Measured in Biochemical System Using Microfluidics - 2046-11_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623979Inhibition of Human CDK5 Activity Measured in Biochemical System Using Microfluidics - 2046-12_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623989Inhibition of Human GSK3 beta Activity Measured in Biochemical System Using Microfluidics - 2046-10_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623998GSK3beta Measured in Biochemical System Using Plate Reader - 2046-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
623999CDK5 Measured in Biochemical System Using Plate Reader - 2046-05_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624027GSK3beta Assay at Varied ATP concentrations Measured in Biochemical System Using Plate Reader - 2046-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624076Kinome profiling single dose point Measured in Biochemical System Using Plate Reader - 2133-09_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
624086TCF/LEF Reporter Assay Measured in Cell-Based System Using Plate Reader - 2133-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624088PathHunter bCatenin (U2-OS) Measured in Cell-Based System Using Plate Reader - 2133-06_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624091Dose responses of selected compounds against GSK3b Measured in Biochemical System Using Plate Reader - 2133-10_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
624108GSK3beta Surface Plasmon Resonance (SPR) Assay Measured in Biochemical System Using Microfluidics - 2133-08_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
Description:
Keywords:
Human Tau, phospho-Tau, (Ser199), SH-SY5Y human neuroblastoma cells, GSK-3b

Assay Overview:
The glycogen synthase kinase-3 beta (GSK-3b) is a known master regulator for several cellular pathways and plays a critical role in metabolism, transcription, development, cell survival, and neuronal functions. The overall objective is to identify one or multiple series of binders of GSK-3beta with sub-micromolar potency. Such compounds will become probe(s) with demonstrated kinase-selectivity. The pTau and total Tau ELISA quantifies the level of activation of Tau protein (phosphorylation at Serine 199), a direct downstream target of GSK-3b. This assay assesses the decrease in GSK-3b activity brought upon by treatment of SH-SY5Y neuroblastoma cells with these compounds. The compounds were tested in a dose-response manner at 9 concentrations in biological duplicates. The value achieved in the pTau ELISA is normalized to the value achieved in the total Tau ELISA to correct for any cell toxicity and plating discrepancies, so eliminate false positive results. A positive control (CHIR99021 at 10 uM) was included in each plate as well as DMSO as a neutral control.
Normalizing the values achieved for total Tau to the values for the DMSO controls across the dose-response curve can also be used as a proxy to determine at which concentration the compound causes cell toxicity.

Expected Outcome:
Quantify total Tau and phospho-Tau using immunoassay kit. Measure inhibiton of GSK3beta by normalizing levels of p-Tau to total Tau
Inhibitors will result in decrease of signals.
Protocol
Human Tau and phospho-Tau (Ser199) Cell-based Immunoassay (ELISA)
Outline:
A standard protocol using Immunoassay kits to quantify total Tau protein and phospho-Tau (Ser199) protein in SH-SY5Y human neuroblastoma cells. Protocol will need to be re-optimized for other cell types as far as plating density and readout within the linear range of the standards.
Tissue Culture:
Media: DMEM 4.5g glucose, L-glut, - sodium pyruvate (cellgro #10-017-CV) / 10% FBS (Invitrogen #10082) / 1% penicillin-streptomycin (Invitrogen #15140)Plate Type: Costar 96 well black, TC treated culture plates ( #8795BC), can also use white or clear Eppendorf 96 well PCR plates, colorless (#951020401)
Reagents:
1. Human Tau (Total) ELISA Kit from Invitrogen (#KHB0041)
a. Human Tau (Total) Standard
b. Standard Diluent Buffer
c. Tau Antibody Coated Wells
d. Human Tau (Total) Detection Antibody
e. Anti-Rabbit IgG HRP
f. Streptavidin-HRP Diluent
g. Wash Buffer Concentrate
h. Stabilized Chromogen
i. Stop Solution
2. Human pTau (Ser199) ELISA Kit from Invitrogen (#KHB7041)
a. Human pTau (Ser199) Standard
b. Standard Diluent Buffer
c. Tau Antibody Coated Wells
d. Human pTau (Ser199) Detection Antibody
e. Anti-Rabbit IgG HRP
f. Streptavidin-HRP Diluent
g. Wash Buffer Concentrate
h. Stabilized Chromogen
i. Stop Solution
Protocol:
1. Seed 50,000 cells in 200 ul per well in cell culture media (250,000 cells/ml)
2. Incubate cells overnight @ 37 degrees C
3. Induce cells with appropriate doses of GSK3 inhibitors
4. Incubate for approximately 24 hrs @ 37 degrees C
5. Harvest cell lysate:
a. Wash cells once with cold PBS
b. Add 100ul cold lysis buffer and pipette up and down vigorously
c. Transfer lysate to 96 well PCR plate
d. Centrifuge at 4,000rpm for 20 minutes at 4 degree C
6. Reagent Preparation:
a. Reconstitute Human Tau (Total) Standard with 1300ul Standard Diluent Buffer.
i. Make serial dilutions of the standard according to the following table:
Standard:Add:Into:2000 pg/ml1000 pg/ml300 ul of 2000 pg/ml standard300 ul Diluent Buffer500 pg/ml300 ul of 1000 pg/ml standard300 ul Diluent Buffer250 pg/ml300 ul of 500 pg/ml standard300 ul Diluent Buffer125 pg/ml300 ul of 250 pg/ml standard300 ul Diluent Buffer62.5 pg/ml300 ul of 125 pg/ml standard300 ul Diluent Buffer32.1 pg/ml300 ul of 62.5 pg/ml standard300 ul Diluent Buffer0 pg/ml300 ul Diluent BufferEmpty Tube
b. Reconstitute Human pTau (Ser199) Standard with 1730ul of Standard Diluent Buffer. Gently mix. Allow to rest for 10 minutes
i. Make serial dilutions of the standard according to the following table:
Standard:Add:Into:1000 pg/ml500 pg/ml300 ul of 1000 pg/ml standard300 ul Diluent Buffer250 pg/ml300 ul of 500 pg/ml standard300 ul Diluent Buffer125 pg/ml300 ul of 250 pg/ml standard300 ul Diluent Buffer62.5 pg/ml300 ul of 125 pg/ml standard300 ul Diluent Buffer31.2 pg/ml300 ul of 62.5 pg/ml standard300 ul Diluent Buffer15.6 pg/ml300 ul of 31.2 pg/ml standard300 ul Diluent Buffer0 pg/ml300 ul Diluent BufferEmpty Tube
c. Dilute anti-rabbit IgG HRP for both Human Tau (Total) and Human pTau (Ser199) by allowing it to reach room temperature, then use the following table:
# of 8 well Strips:Vol of anti-rabbit IgG HRP:Vol of HRP Diluent:220 ul 2 ml440 ul4 ml660 ul6 ml880 ul8 ml10100 ul10 ml12120 ul12 ml
d. Dilute wash buffer from 25X concentrate:
i. Amount of wash buffer = (x)*(8)*(400)*(4)*(3), where x = number of strips of ELISA plate
ii. Dilute 24 volumes with deionized water (ie 5ml concentrate into 120ml water)
7. Warm well strips and determine how many are needed, insert them into the frame
8. Load standards:
a. Load 100ul of standard dilluent buffer into ZERO wells
b. Load 100ul of each standard concentrations (both total Tau and pTau) to wells in DUPLICATE
9. Load samples:
a. To each well that will be analyzed for total Tau, add 85ul standard diluent buffer + 15ul cell lysat
b. To each well that will be analyzed for pTau (Ser199) add 50ul standard diluent buffer + 50ul cell lysate
c. BE SURE TO NOTE WHICH WELLS/PLATES WILL BE ANALYZED for total Tau versus pTau.
d. Tap gently on side of plate to mix
10. Leave duplicate wells empty for chromogen blank
11. Cover and incubate plate for 2 hours at room temperature
a. NOTE: Maintain same order of addition for all reagents to ensure equal incubation times for all wells
12. Aspirate wells, do not scratch the inside wall of wells
13. Wash wells 4 times:
a. Add 400ul diluted wash buffer, leave for 30 seconds, aspirate all wells
b. Alternatively, use automated plate washer and program in a 30 second hold between each cycle
14. Add 100ul of detection antibody solution to each well EXCEPT chromogen blanks
a. Add anti-total Tau to wells being analyzed for total Tau
b. Add anti-pTau (Ser199) to wells being analyzed for pTau (Ser199)
c. Tap gently on side of plate to mix
15. Cover and incubate plate for 1 hour at room temperature
16. Aspirate and wash wells 4 times as before
17. Add 100ul of anti-rabbit IgG HRP working solution (prepared ahead) to each well EXCEPT chromogen blanks
18. Cover plate and incubate for 30 minutes at room temperature
19. Aspirate and wash wells 4 times as before
20. Add 100ul of stabilized chromogen to each well, the liquid will turn blue
21. Incubate for 30 minutes at room temperature in the DARK, do not cover plate with foil
22. Add 100ul of stop solution to each well, tap gently on side of plate to mix
23. Read the absorbance of each well at 450nm (within 2 hours of adding stop solution)
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: SH-SY5Y
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_uM *The concentration at which activity reaches 50% of the maximumFloatμM
2% Cell Survival_at_11mM_Test1 Percent of cell survival at the tested concentration.Float%
3% Cell Survival_at_35mM_Test1 Percent of cell survival at the tested concentration.Float%
4% Cell Survival_at_11mM_Test2 Percent of cell survival at the tested concentration.Float%
5% Cell Survival_at_35mM_Test2 Percent of cell survival at the tested concentration.Float%
6% Cell Survival_at_11mM_Test3 Percent of cell survival at the tested concentration.Float%
7% Cell Survival_at_35mM_Test3 Percent of cell survival at the tested concentration.Float%

* Activity Concentration.
Additional Information
Grant Number: 1 RO3 MHD87442-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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