Microsoft word - eerk.doc

BioAssay Systems ERK Phoshorylation EERK001.pdf
EnzyFluoTM ERK Phosphorylation Assay Kit (EERK-100)
Fluorimetric Cell-Based Assay for ERK Phosphorylation Status
DESCRIPTION
The mitogen-activated protein kinase (MAPK/ERK) pathway plays a key
2. Treat the cells as desired (e.g. with ligands or drugs). role in cell proliferation, differentiation and migration. Stimulation by 3. Prepare formaldehyde solutions (warning: formaldehyde is toxic. Use mitogens eventually leads to phosphorylation of ERK1 (T202/Y204) and chemical hood and wear appropriate gloves and eye protection): ERK2 (T185/Y187). The MAPK/ERK cascade presents many interesting drug targets for the development of cancer therapies. For adherent cells, prepare 4% formaldehyde by mixing 1.3 mL of 37% formaldehyde and 10.7 mL of 1× Wash buffer. Simply fix cells in each BioAssay Systems' cell-based ELISA measures dually phosphorylated ERK1/2 in whole cells. This simple and efficient assay eliminates the need well by replacing the medium with 100 µL of 4% formaldehyde. for cell lysate preparation and can be used to study kinase signaling and For suspension cells, prepare 8% formaldehyde by mixing 2.6 mL of 37% the effects of kinase inhibitors on cells. In this assay, cells are grown in 96- formaldehyde and 9.4 mL of 1× Wash buffer. Centrifuge the plate at 500g well plates and treated with ligands or drugs. Cells are then fixed and for 15 min at 4°C and carefully remove as much media as possible permeabilized in the wells. ERK1/2 phosphorylation (pERK) is measured without disturbing the cell pellet (repeat this for suspension cells with using a double immunoenzymatic labeling procedure. each wash step below). Fix the cells in each well by adding 100 µL of 8% Principle of Cell-Based ERK ELISA
Cover the plate and incubate for 20 min at room temperature. HRP (535/590nm)
ALP (360/450nm)
Alternatively, the plate containing the fixed cells can be sealed and 4. Remove the formaldehyde solution and wash the cells 3 times with 150 A. Culture &
B. Add Ab1
C. Add Ab2
D. Add fluorogenic
treat cells
µL of 1× Wash Buffer. Each wash step should be performed for 5 min substrates. Measure
fluorescence intensity.
Primary Ab1:
5. Prepare Quench Buffer by mixing 2.2 mL of 3% H Secondary Ab2:
KEY FEATURES
Remove the Wash Buffer and add 100 µL of Quench Buffer to each assay well. Cover plate and incubate for 20 min at room temperature. Simple and convenient. Total and pERK can be measured in the same 6. Remove the Quench Buffer and wash the cells 3 times with 150 µL of 1× Wash Buffer. Each wash step should be performed for 5 min with gentle APPLICATIONS
Determination of ERK phosphorylation status in whole cells. 7. Remove the Wash Buffer, and add 100 µL of Blocking Buffer. Cover Evalutation of effects of ligands or drugs on ERK phosphorylation. plate and incubate for 1 hr at room temperature. Species tested: human, mouse, rat.
KIT CONTENTS
B. Add Primary Antibodies (Ab1)
1. Add 100 µL of PBS to the ERK-Ab1 and pERK-Ab1 tubes and mix well. 10× Wash Buffer: 25 mL
Blocking Buffer: 25 mL
Prepare enough primary antibody Ab1 Mixture for each well by mixing 1 ALP Substrate:
HRP Substrate: 6 mL
µL diluted ERK-Ab1, 1 µL diluted pERK-Ab1 and 55 µL Blocking Buffer. ERK-Ab1:
pERK-Ab1
Unused Ab1 antibodies can be stored at -20°C for up to 45 days. HRP-Ab2:
Storage conditions: store all reagents at -20°C. Shelf life of 6 months after
2. Remove the Blocking Buffer from all assay wells. Add 50 µL of the Blocking Buffer to the Sample Background wells and 50 µL of Ab1 Precautions: reagents are for research use only. Normal precautions for
Mixture to the Sample wells. Cover plate and incubate for 3 hrs at room laboratory reagents should be exercised while using the reagents. Please temperature or overnight at 2-8°C with gentle shaking. refer to Material Safety Data Sheet for detailed information. 3. Remove the Ab1 Mixture and wash the cells 3 times with 150 µL of 1× Wash Buffer. Each wash step should be performed for 5 min with gentle ASSAY PROCEDURE
1. To avoid cross-contamination, change pipette tips between additions of C. Add Secondary Antibodies (Ab2)
each reagent or sample. Use separate reservoirs for each reagent. Prior 1. Immediately before use, add 100 µL of PBS to the HRP-Ab2 and ALP- to Assay, dilute 10× Wash Buffer in dH2O to prepare 250 mL 1× Wash Ab2 tubes and mix well. Prepare enough secondary antibody Ab2 Mixture, for each well, by mixing 1 µL diluted HRP-Ab2, 1 µL diluted 2. It is recommended that assays be run in duplicate. Plan to use four ALP-Ab2 and 55 µL Blocking Buffer. Unused Ab2 antibodies can be assay wells for each sample: two "Sample Background" wells in which Blocking Buffer is added for determining Ab2 background fluorescence, and two "Sample" wells in which Ab1 Mixture is added (see Step B2). 2. Remove Wash Buffer and add 50 µL of the Ab2 Mixture to all assay wells. Cover plate and incubate for 2 hrs at room temperature with gentle shaking. A. Culture and Treat Cells
1. Seed 100 µL of 2-4 × 104 adherent cells (or 4-10 × 104 suspension cells) into each well of a black clear-bottom 96-well plate. Incubate overnight at D. Detection
1. Remove the Ab2 Mixture from each well and wash the cells 5 times with 150 µL of 1× Wash Buffer. Each wash step should be performed for 5 Note: The cell number to be used depends on the cell line and ERK1/2 2012  by BioAssay Systems · 3191 Corporate Place, Hayward, CA 94545, USA · Website: www.bioassaysys.com Tel: 510-782-9988, Fax: 510-782-1588 · Email: [email protected], [email protected] BioAssay Systems ERK Phoshorylation EERK001.pdf
2. Immediately before use, add 6 µL 3% H2O2 to the provided 6 mL HRP Substrate (for partial plate assay, adjust the volumes accordingly). pERK/PMA
pERK/Inhibitor
Remove the Wash Buffer from the plate and add 50 µL of reconstituted HRP Substrate to each well. Incubate for 20 min at room temperature in 3. Add 50 µL of ALP Substrate to each well and incubate for an additional 4. Read the plate at λex/em = 535/590nm for phosphorylated ERK (pERK) and at λex/em =360/450nm for total ERK (ERK). Time (min)
[Sorafenib], M
Left: Kinetics of ERK1/2 phosphorylation in PANC-1 cells on treatment with
CALCULATION
phorbol myristate acetate (PMA). Right: inhibition of ERK1/2 phosphorylation by
the kinase inhibitor Sorafenib. Cells were treated with drug for 3 hours and then 5
Calculate the mean fluorescence intensities for the Sample Background wells and Sample wells. Subtract the mean fluorescence of the Sample 50 values were 2.1, 11.4 and 11.5 µ M respectively, for RBL-243, Background wells from the fluorescence value of the Sample well to yield ∆F values for the phosphorylated ERK (∆FpERK) at 535/590nm and the total
ERK (∆FERK) at 360/450nm.
LITERATURE
1. Cobb MH, et al (1994). The mitogen-activated protein kinases, ERK1 and Normalized phosphorylated ERK (pERK) is calculated as, 2. Daniluk J, Dabrowski A. (2007). The effect of concomitant stimulation ∆FPERK /∆FERK
with cholecystokinin and epidermal growth factor on extracellular signal- Normalized pERK =
regulated kinase (ERK) activity in pancreatic acinar cells. J Physiol PERK /∆FERK)O
where (∆FpERK / ∆FERK)o is the control reference value (e.g. time zero in
3. Iqbal J, et al (2007). Rapid in vivo effects of estradiol-17beta in ovine kinetic studies or untreated wells in drug potency studies.) pituitary gonadotropes are displayed by phosphorylation of extracellularly regulated kinase, serine/threonine kinase, and 3',5'-cyclic adenosine 5'- MATERIALS REQUIRED BUT NOT PROVIDED
monophosphate-responsive element-binding protein. Endocrinology 148 (12): 5794-802. 37% formaldehyde (Sigma, cat # F8775); 3% H2O2 (Sigma, cat # H1009); black cell culture 96-well plate: available separately at BioAssay System (cat# AB96WP) or at VWR (cat# 82050-748); plate sealers: available separately at BioAssay Systems (cat# AB96SL) or at Sigma (cat# A5596); deionized or distilled water; pipetting devices; cell culture incubators; centrifuge tubes; fluorescence plate reader capable of reading at λex/em = 535/590nm and at λex/em =360/450nm. Example of A 96-Well Assay Plate Lay-out
2012  by BioAssay Systems · 3191 Corporate Place, Hayward, CA 94545, USA · Website: www.bioassaysys.com Tel: 510-782-9988, Fax: 510-782-1588 · Email: [email protected], [email protected]

Source: http://www.totallab.co.nz/productfiles/BioAssay%20Systems/EERK-100.pdf