Detection of Phospho-Hsp27 in THP.1 cells by Bioplex Assay

Friday, June 11, 2004

Description
Medium throughput assay for detection of the phosphorylation of Hsp27 by upstream kinases (notably MapKap Kinase 2)

Procedure
Phospho-HSP27 Cellular Assay

Cell Type:
THP-1 cells from ATCC (TIB-202) that have been serum-starved (0.5% FBS) overnight

Source and Composition of test agents:
Drug lead compounds or controls are made-up in 100% DMSO by the in-house Pharmacy.

Dilution Scheme:
In a 96-well dilution plate (or plates), compounds are serially diluted 1:5 with 100% DMSO.
Into wells of dilution plate(s), (i.e. row A) apply ~100 uL/well of initial stock. Then, in next well (i.e. row B) apply 80uL/well of DMSO + 20uL/well of the previous dilution for 1/5 dilution. Mix by pipetting up and down several times. Repeat serial dilution to give a total of 6 assay points per compound (rows A-F).
Rows G and H contain only DMSO for 100% and blank control values.

Compounds are directly transferred to a daughter plate to create a 4X test plate in low serum RPMI media (0.5% FBS)

In a 0.8ml 96-well storage plate(s), directly transfer 10uL/well of each drug or control dilution in DMSO to the corresponding well in the storage plate containing 500uL of low serum RPMI media.

Set Up of Test Plate:
1) Seeding of cells
Serum-starved THP-1 cells are seeded to the appropriate number of 96 well Multi-screen filter plates (1 plate per 5 test compounds) at a density of 2 x 105/well in 100uL of low serum RPMI media (0.5% FBS)

2) Addition of test compounds
Directly transfer 50uL from the storage plate to the test plate (in duplicate). See ATTACHED plate template for the layout.

Pre-Incubation:
Cells and compounds or controls are pre-incubated for 1 hour in a 370C incubator.

Induction of Cytokine:
1) Preparation of Inducing agent:
LPS is diluted from a 500 ug/ml stock solution made in endotoxin free dIH2O, aliquot, and stored at 200 C.

2) Preparation of working stock:
Thaw and dilute an aliquot of the 500ug/ml LPS stock 1:125 with low serum (0.5% FBS) RPMI media (final 4ug/ml) and add 50uL/well to all wells, except to negative control wells. Mix by carefully pipetting (100uL) up and down several times. LPS final concentration is 1mg/ml per well, excepting negative control wells (row H).

3) Incubate plates in an incubator set at 370C for 45 mins.

Collection of Samples for phospho-HSP27 Analysis:
After incubation, plates are vacuum filtered to remove media and compounds. Cells are washed twice with Assay Buffer #1 using vacuum filtration. Then, 100 uL of cell lysis buffer is added per well and plate is covered and shaken for 20mins at 4oc to lyse cells. Lysates are directly transferred to a flat bottom 96 well plate for analysis or stored frozen at 20oc until analysis.

Analysis of phospho-HSP27 via Bio-Plex:
1) Preparation of cell lysates
Thaw lysates (if frozen) and dilute 1:2 with assay buffer #1

2) Preparation of Beadmates/96 well plate
Vortex the phospho-HSP27 Beadlyte capture beads for 15secs then sonicate for an additional 30secs. Dilute beads to 1X by adding the entire contents (125ul) of capture beads to 2.475ml of cell assay buffer #1

3) Preparation of Reporter/96 well plate
Dilute the reporter to 1X by adding the entire contents (125ul) to 2.475ml of cell assay buffer #1

4) Preparation of Streptavidin-Phycoerythrin/96 well plate
Dilute streptavidin-phycoerythrin to 1X by adding 50ul to 2.5ml of cell assay buffer #1 (1:50)

5) Main protocol
a. Pre-wet 96well filter plate with 25ul assay buffer #1. Remove by vacuum filtration.
b. Quickly vortex and sonicate 1X bead suspension and add 25ul/well
c. Add 25ul diluted lysate/well. Cover with plate sealer and wrap in aluminum foil. Incubate overnight at 4oc on a plate shaker.
d. Remove lysate by vacuum filtration. Wash beads with 100ul assay buffer #1 and vacuum filter. Gently blot bottom of plate to remove excess liquid.
e. Add 25ul of 1X reporter/well. Cover and re-wrap the plate in foil. Incubate for 1hr at RT on a plate shaker.
f. Remove reporter by vacuum filtration and blot bottom of the filter plate to remove excess liquid.
g. Add 25ul 1X streptavidin-phycoerythrin/well. Cover and re-wrap plate in foil. Incubate for 30mins at RT on a plate shaker.
h. Remove streptavidin-phycoerythrin by vacuum filtration and blot the bottom of the filter plate to remove excess liquid.
i. Resuspend beads in 125ul assay buffer #1. Cover and re-wrap in foil. Shake for 1min before reading plate Bio-Plex

Recipes

Supplies
Reagent Vendor Catalog Number
Dimethyl Sulphoxide (DMSO) Sigma D2650
RPMI Media Gibco 11875-093
Endotoxin free dIH2O Gibco 15230-170
Foil Plate Sealers Beckman 538619
96-well storage plates (polypropylene) Abgene AB-0765
96-well Multiscreen-GV plates Millipore MAGVS2210
96-well flat bottom plates Corning 3599
Cell Lysis Kit Biorad 171-304011
Lipopolysaccharide Escherichia (LPS) Sigma L-4516
Phospho-HSP27 Beadmates UBI 46-607
Cell Assay Buffer #1 UBI 43-010
Streptavidin-Phycoerythrin UBI 45-001

Tips
can also use a detection kit from Biosource. Spot check with using bioplex on total Hsp27 protein to make sure you are preparing samples evenly...this is not necessary for every assay.

(责任编辑：泉水)

搜索

热门标签:

Detection of Phospho-Hsp27 in THP.1 cells by Bioplex Assay