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Ppargamma Elisa Kit (Chemiluminescence) : Signosis

This document describes a PPARgamma ELISA kit from Signosis that allows for the sensitive and specific measurement of PPARgamma activation. The kit includes a 96-well plate coated with a PPARgamma consensus sequence and antibodies to detect activated PPARgamma bound to the plate. The procedure involves incubating nuclear extract or cell lysate samples in the plate, washing, incubating with detection antibodies, washing again, and detecting chemiluminescence to quantify activated PPARgamma levels. The kit provides the necessary buffers, controls, and substrates to perform the assay according to the outlined protocol.

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Nyayu Fitriani Muttaqien
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40 views2 pages

Ppargamma Elisa Kit (Chemiluminescence) : Signosis

This document describes a PPARgamma ELISA kit from Signosis that allows for the sensitive and specific measurement of PPARgamma activation. The kit includes a 96-well plate coated with a PPARgamma consensus sequence and antibodies to detect activated PPARgamma bound to the plate. The procedure involves incubating nuclear extract or cell lysate samples in the plate, washing, incubating with detection antibodies, washing again, and detecting chemiluminescence to quantify activated PPARgamma levels. The kit provides the necessary buffers, controls, and substrates to perform the assay according to the outlined protocol.

Uploaded by

Nyayu Fitriani Muttaqien
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Signosis

Innovative Plate Assay Solutions

PPARgamma ELISA Kit (Chemiluminescence)


Catalog Number TE-0010 (For Research Use Only)

Introduction
Peroxisome-proliferator-activated receptor gamma (PPAR
gamma) is a ligand-activated transcription factor that plays
an important role in the control of gene expression
associated with a variety of physiological processes,
particularly in metabolism and adipogenesis. With ligand
binding, PPAR gamma forms a heterodimer with the
retinoic X receptor, and binds to PPAR response elements
in the promoter region of target genes, thus regulating their
expression. Dysfunction of PPARgamma leads to the
pathological processes, such as metabolic diseases and
cancer. Furthermore, modulation of receptor action in
these diseases may be of therapeutic value. PPARgamma
has been demonstrated to be the target of the
thiazolidinediones (TZDs), which are widely used to treat
type 2 diabetes. Signosis has developed PPARgamma
ELISA, to help specific and sensitive measurement of the
activation of PPARgamma.

Principle of the assay


Diagram of TF ELISA
PPARgamma ELISA kit is highly sensitive and specific
assay with a simple and optimized procedure. The 96-well Materials provided with the kit
(8X12 strip) white plate is pre-immobilized with the
PPARgamma consensus sequencing oligo. The activated  96 well microplate coated with PPARgamma
PPARgamma in nuclear extract or the whole cell lysate is consensus oligo (4oC)
added in the well and binds to the oligo. The activated  Antibody against PPARgamma (4oC)
PPARgamma is detected with a specific antibody against  HRP conjugate secondary antibody (4oC)
PPARgamma subunit and a HRP conjugated secondary
 2X TF binding buffer (-20oC)
antibody. The assay utilizes chemiluminescence detection
 1X Nuclear extract dilution buffer (-20oC)
method, which can be easily measured by
spectrophotometry.  1X Diluent buffer (4oC)
 5X Assay wash buffer (4oC)
 Positive control (-20oC)
 Substrate A (4oC)
 Substrate B (4oC)
 Substrate dilution buffer (4oC)

Material required but not provided


 Luminometer
 Deionized or distilled water

Signosis, Inc. • 1700 Wyatt Drive Suite 10-12 • Santa Clara, CA 95054 • Tel 408 747 0771 • Fax 408 864 2182
10. Add 95µl of substrate solution to each well.
Reagent preparation before starting 11. Place the plate into a luminometer and incubate it
experiment inside the reading chamber (in complete darkness)
for 2 minutes. Set integration time to 1 second
 Dilute the 5X Assay wash buffer to 1X Assay wash with no filter position. For the best results, read
buffer by combining the following reagents: the plate within 5-20 minutes.
40ml 5X Assay wash buffer
160ml ddH2O
 Dilute 500 times of antibody against PPARgamma
with 1X Diluent buffer before use.
 Dilute 1000 times of HRP conjugate secondary
antibody with 1X Diluent buffer before use.

Assay procedure
1. Cut the sealing film over the plate and remove it
from the desired number of well strips. Make sure
the rest of wells are well sealed.
2. Prepare TF binding mix by combining the following
reagents:
25ul 2X TF binding buffer
X Nuclear extract (2-10ug)
X Nuclear extract dilution buffer
Total 50ul
Note: For positive control, use 25ul of
positive control without adding unclear
extract dilution buffer.
3. Add the mix on a well and incubate for 1 hour with
gently shaking at room temperature.
4. Discard the contents and wash by adding 200µl of
1X Assay wash buffer. Repeat the process three
times for a total of three washes. Completely
remove liquid during each wash by inverting the
plate against clean paper towels. After the last wash,
ensure all the liquid has been removed in each well.
5. Add 100µl of diluted antibody against PPARgamma
to each well and incubate for 1 hour at room
temperature with gentle shaking.
6. Repeat the aspiration/wash process as instructed in
step 4.
7. Add 100µl of diluted HRP conjugate secondary
antibody to each well and incubate for 45 min at
room temperature with gentle shaking.
8. Invert the plate over an appropriate container and
expel the contents forcibly. Wash the plate by adding
200µl of 1X Assay wash buffer. Incubate the plate
with wash buffer for 10 minutes on a shaker. Repeat
aspiration/wash process two times for a total of three
washes with 10 minutes 1X Assay wash buffer
incubation between each wash. Completely remove
liquid at each wash by firmly tapping the plate
against a pile of clean paper towels.
Note: It is important to incubate the plate with
wash buffer for 10 minutes during each wash to
reduce high background in the blank wells.
9. Prepare the substrate solution for the whole plate by
combining the following reagents:
1ml Substrate A
1ml Substrate B
8ml Substrate dilution buffer

Signosis, Inc. • 1700 Wyatt Drive Suite 10-12 • Santa Clara, CA 95054 • Tel 408 747 0771 • Fax 408 864 2182

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