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BioAssay: AID 588510

Fluorescence Polarization Biochemical Secondary LANA-Nucleosome Assay_2053_04_Inhibitor_Dose_DryPowder_Activity

This assay uses a different method to confirm that the compounds specifically inhibit the binding of LANA to the histones (present in the purified nucleosomes). Nucleosomes are bound to the microtiter plate, followed by the addition of GST-tagged N-terminal LANA 1-23 peptide. Mitoxantrone, a known DNA intercalator, will be used as the positive control because it is known to disrupt the binding of LANA to the histones, H2A & H2B. ..more
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 Tested Compounds
 Tested Compounds
All(13)
 
 
Inactive(13)
 
 
 Tested Substances
 Tested Substances
All(13)
 
 
Inactive(13)
 
 
AID: 588510
Data Source: Broad Institute (2053_04_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2011-10-13

Data Table ( Complete ):           All
Target
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
2659Summary of Broad Institute MLPCN Kaposi's Sarcoma Herpes Virus Latent Infection ProjectsummarySummary of Broad Institute MLPCN Kaposi's Sarcoma Herpes Virus Latent Infection Project.
Description:
Keywords: LANA, KSHV, ELISA,

Assay Overview:
This assay uses a different method to confirm that the compounds specifically inhibit the binding of LANA to the histones (present in the purified nucleosomes). Nucleosomes are bound to the microtiter plate, followed by the addition of GST-tagged N-terminal LANA 1-23 peptide. Mitoxantrone, a known DNA intercalator, will be used as the positive control because it is known to disrupt the binding of LANA to the histones, H2A & H2B.

Expected Outcome:
The majority of compounds should inhibit binding in a dose dependent manner. It is possible that some compounds will not inhibit the binding below an IC50 of 10 uM. Since this is not a flourescence assay, it will also eliminate any influence of autofluorescence or quenching on the assay which may have influenced the outcome in the primary assay using fluorescence polarization.
Protocol
Protocol:

LANA-Nucleosome 96 Well ELISA Protocol

1.Add purified nucleosomes 1.5 ug/well of 96 well plate in 100 uL PBS (Use Immuolon HB plates). Incubate O/N at 4 degrees C
2.Wash 3x with 150 ul PBS/0.05% Tween-20
3.Add 30 uL PBS/5% milk & incubate at RT for 1 hour
4.Wash 3x 150 ul with PBS/0.05% Tween-20
5.Add GST-LANA 1-23 at 7.5 ug/mL in 100 uL PBS/0.2% Tween/1% milk.
6.Pin with compound (or provide poscon in part of plate in step 5). Incubate for 90 minutes at 4 degrees C.
7.Wash 3x with 150 ul PBS/0.05% Tween-20
8.Add anti-GST primary antibody at 1:2000 in 100 uL PBS/0.2% Tween/2% milk. Incubate 90 minutes at 4 degrees C.
9.Wash 3x with 150 ul PBS/0.05% Tween-20
10.Add secondary antibody at 1:5000 in 100 uL PBS/0.2% Tween/2% milk. Incubate 90 minutes at 4 degrees C.
11.Wash 3x with 150 ul PBS/0.05% Tween-20
12.Add Sigma Fast substrate, 50 uL per well in water. Develop the plate in the dark for 30 minutes at RT.
13.Read plates at 450 nM. (If the plate will not be read immediately, add 15 uL of 3M HCl or 3M H2SO4 per 50 ul solution and read at 492 nm.

PBS/5% milk (for 2 plates):
5 mL 10x PBS
2.5 g Bio-Rad blotting grade dry milk
45 mL water

PBS/0.2% Tween-20/1% milk:
5 mL 10% Tween-20
25 mL 10x PBS
2.5 g Bio-Rad blotting grade dry milk
220 mL water

PBS/0.2% Tween-20/2% milk:
5 mL 10% Tween-20
25 mL 10x PBS
5 g Bio-Rad blotting grade dry milk
220 mL water


ELISA Wash Buffer (PBS/0.05% Tween-20)
5 mL 10% Tween-20
1 L PBS

Nucleosomes @ 31.5 uM (13.86 ug/uL)
For 1 96 well plate, dilute 52.8 uL into 12 mL PBS to coat

GST-LANA 1-23 stock @8.9 ug/uL
For 1 plate, dilute 10.1 uL GST-LANA to 12 mL PBS/0.2% Tween/1% milk
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1AC_50uM*FloatμM
2Activity_at_0.016uM_(%)The average measured activity of all accepted replicates at the specified concentrationFloat%
3Activity_at_0.08uM_(%)The average measured activity of all accepted replicates at the specified concentrationFloat%
4Activity_at_0.4uM_(%)The average measured activity of all accepted replicates at the specified concentrationFloat%
5Activity_at_2uM_(%)The average measured activity of all accepted replicates at the specified concentrationFloat%
6Activity_at_10uM_(%)The average measured activity of all accepted replicates at the specified concentrationFloat%
7Activity_at_50uM_(%)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration.
Additional Information
Grant Number: 1 R21 NS061738-01

Data Table (Concise)
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