Product name :EnzyFluo™ AMPK Phosphorylation Assay Kit

Catalog Number :EAMPK-100

Quantity :100 tests

Price :373 Eur

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Supplier :Bioassays

DESCRIPTION
The 5-AMP-activated protein kinase (AMPK) is a key sensor of intracellular
energy balance. AMPK is activated in response to an increase in the
AMP/ATP ratio which can be caused by a number of factors such as
muscle contraction, starvation, or hypoxia. AMPK is a heterotrimeric protein
complex comprising of α- (63 kDa), β- (38 kDa) and γ- (38 kDa) subunits.
For each subunit, isoforms have been identified (α1, α2, β1, β2, γ1, γ2, γ3)
which theoretically allow the formation of 12 different proteins. The α-
subunit contains a serine/threonine kinase domain and the regulatory
subunits contain binding sites for AMP and ATP (γ-subunit) and for
glycogen (β-subunit).
AMPK is activated by phosphorylation on Thr-172 within the catalytic
domain. AMP binding results in a 2 to 5-fold increase in AMPK activity
compared to the basal level. Binding of AMP to the γ-subunit causes
allosteric activation of the kinase and induces a conformational change in
the kinase domain that protects AMPK from dephosphorylation of Thr-172.
BioAssay Systems' cell-based ELISA measures phosphorylated AMPK in
whole cells and normalizes the signal to the total protein content. The
antibody recognizes both α-subunits and thus can be used for cells from all
tissues (human, mouse, rat). This simple and efficient assay eliminates the
need for cell lysate preparation and can be used to study AMPK regulation
in short-term and long-term assays. In this assay, cells grown in 96-well
plates are fixed and permeabilized in the wells. AMPK phosphorylation
(pAMPK) is measured using a fluorescent ELISA followed by total protein
measurement in each well.
Principle of Cell-Based AMPK ELISA
A. Culture &
treat cells
B. Add Ab1 C. Add Ab2 D. Add fluorogenic
substrates. Measure
fluorescence intensity. rabbit anti-phospho AMPK
anti-rabbit IgG-HRP
Primary Ab1:
Secondary Ab2:
Protein (360/450nm)
pAMPK AMPK pAMPK AMPK
Y
pAMPK AMPK
Y
pAMPK AMPK
Y
HRP (530/585nm)
Protein Stain:


KEY FEATURES
Sensitive. Can measure pAMPK modulation in as little as 500 cells/well.
Simple and convenient. No cell lysis necessary, cells can be cultured for
several days.


APPLICATIONS
Determination of AMPK phosphorylation status in whole cells.
Evaluation of direct and indirect modulation of AMPK phosphorylation.
Species tested: human, mouse, rat.


KIT CONTENTS
10x Wash Buffer: 25 mL Blocking Buffer: 25 mL
Protein Stain: 6 mL HRP Substrate: 6 mL
pAMPK-Ab1 10 μL HRP-Ab2 10 μL
Storage conditions: this kit is shipped on ice. Upon delivery, store all
reagents at -20°C. Shelf life of 6 months after receipt.
Precautions: reagents are for research use only. Normal precautions for
laboratory reagents should be exercised while using the reagents. Please
refer to Material Safety Data Sheet for detailed information.

MATERIALS REQUIRED BUT NOT PROVIDED
37% formaldehyde (Sigma, cat # F8775); 3% H2O2 (Sigma, cat # H1009);
black (clear bottom) cell culture 96-well plate: available separately at
BioAssay Systems (cat# P96BCC) or at Sigma (cat# CLS3603); 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 lex/em = 530/585nm and at lex/em =360/450nm.

datasheet of product :EnzyFluo™ AMPK Phosphorylation Assay Kit EnzyFluo™ AMPK Phosphorylation Assay Kit

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