Sequence: LANA [Human herpesvirus 8]

Gene:HHV8GK18_GP81     Conserved Domain     Related Protein 3D Structures

AID	Name	Type	Comment
2659	Summary of Broad Institute MLPCN Kaposi's Sarcoma Herpes Virus Latent Infection Project	summary	Summary of Broad Institute MLPCN Kaposi's Sarcoma Herpes Virus Latent Infection Project.

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:

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

Grant Number: 1 R21 NS061738-01