Protocol for AlphaScreen SureFire 4E-BP1 (p-Thr37

Protocol for AlphaScreen SureFire 4E-BP1 (p-Thr37
AlphaScreen® SureFire®
4E-BP1 (p-Thr37/46) Assay Kits
Quick Guide
Assay Points
500
10 000
50 000
Catalog #
TGR4ES500
TGR4ES10K
TGR4ES50K
For Research Use Only
Research Reagents for Research Purposes Only
TGRKVS61.6
24 July 2015
SureFire is a registered trademark of TGR BioSciences Pty Ltd, Australia. PerkinElmer, Proxiplate, OptiPlate and AlphaScreen are registered trademarks of PerkinElmer, Inc.
page 1
General Information on the AlphaScreen® SureFire® 4E-BP1 p-Thr37/46 assay
The AlphaScreen® SureFire® 4E-BP1 p-Thr37/46 assay is used to measure the phosphorylation of
endogenous 4E-BP1 in cellular lysates. The assay is an ideal system for the screening of small
molecule inhibitors of upstream signaling events, and can be applied to primary cells.
This assay eliminates the need for laborious techniques, such as Western blotting or conventional
ELISA. It is a homogeneous assay, in that no sample washing steps are required, which allows for
minimal handling, short assay times, and robotic operation if desired. The assay utilizes the beadbased Alpha Technology, and requires an Alpha Technology-compatible plate reader.
Alpha Technology AlphaScreen® SureFire® Assay Principle
AlphaScreen® SureFire® technology allows the detection of phosphorylated proteins in cellular
lysates in a highly sensitive, quantitative and user friendly assay. In these assays, sandwich
antibody complexes, which are only formed in the presence of analyte, are captured by
AlphaScreen donor and acceptor beads, bringing them into close proximity. The excitation of the
donor bead provokes the release of singlet oxygen molecules that triggers a cascade of energy
transfer in the Acceptor beads, resulting in the emission of light at 520-620nm.
Kit-Specificity information
This assay kit contains antibodies which recognize the phospho-Thr37/46 epitope, and a distal
epitope, on 4E-BP1. The protein detected by this kit corresponds to GenBank Accession NP_004086.
4E-BP1 is also known as BP-1, 4EBP1; PHAS-I, MGC4316 and EIF4EBP1.
These antibodies recognize 4E-BP1 of human, mouse and rat origin. Other species should be tested
on a case-by-case basis.
TGRKVS61.6
24 July 2015
SureFire is a registered trademark of TGR BioSciences Pty Ltd, Australia. PerkinElmer, Proxiplate, OptiPlate and AlphaScreen are registered trademarks of PerkinElmer, Inc.
page 2
Kit Contents
Kit Size
500 points
10,000 points
50,000 points
Lysis buffer (5X)
1 x 10 mL
4 x 60 mL
3 x 400 mL
Activation buffer
1 x 2 mL
1 x 60 mL
1 x 300 mL
Reaction buffer
1 x 2.6 mL
1 x 45 mL
1 x 225 mL
Dilution buffer
1 x 1.5 mL
1 x 25 mL
2 x 60 mL
Positive Control Lysate
1 tube to be re-dissolved in 250 µL H 2 O
Negative Control Lysate
1 tube to be re-dissolved in 250 µL H 2 O
Storage conditions upon receipt
The kit buffers e.g. 5X Lysis buffer, Activation buffer and Reaction buffer should be stored at 4°C.
DO NOT freeze the kit buffers – the Reaction buffer contains antibodies and freeze/thaw cycles can
lead to a loss of activity.
Materials Required But Not Provided
The AlphaScreen SureFire assay kits are optimized to work with AlphaScreen Protein A general IgG
detection beads. These are available separately from PerkinElmer. The AlphaScreen Protein A
general IgG detection kits contain a biotinylated rabbit IgG control, which can be used to test the
instrument settings and bead performance.
Item
Suggested
source
Protein A general IgG detection kit
(contains the Acceptor and Donor Beads)
PerkinElmer Inc.
Proxiplate™-384 Plus, white, shallow well
assay plate
PerkinElmer Inc.
Optiplate™-384 Plus, white, assay plate
PerkinElmer Inc.
TopSeal-A 384, clear adhesive sealing film
Envision® or Enspire® Alpha-reader
PerkinElmer Inc.
PerkinElmer Inc.
TGRKVS61.6
Catalog #
Size
6760617C
6760617M
6760617R
6008280
6008289
6007290
6007299
6050185
-
500 pt
10,000 pt
50,000 pt
50/box
200/box
50/box
200/box
100/box
-
24 July 2015
SureFire is a registered trademark of TGR BioSciences Pty Ltd, Australia. PerkinElmer, Proxiplate, OptiPlate and AlphaScreen are registered trademarks of PerkinElmer, Inc.
page 3
Buffer preparation and subsequent storage conditions
Store 5X Lysis buffer at 4°C. For assay, dilute 5-fold in
water immediately prior to use. Discard unused buffer.
5X Lysis buffer
Reaction buffer*
Precipitation will occur during storage 4°C. To redissolve, warm to 37°C and mix. Alternatively, Activation
buffer can be stored at room temperature with no loss
in activity.
Keep on ice while in use. Do not freeze.
Once diluted discard unused reaction buffer.
AlphaScreen® Protein A IgG Kit
Store at 4°C in the dark.
Activation buffer
Immediately prior to use, dilute Activation buffer
5-fold in Reaction buffer (e.g. take 98 μL Activation
buffer and dilute in 392 μL Reaction buffer).
Acceptor Mix
(Reaction buffer + Activation buffer +
AlphaScreen® Acceptor beads)
Donor Mix**
(Dilution buffer + AlphaScreen®
Donor beads)
Dilute Acceptor beads 50-fold in Acceptor mix (e.g. add
10 μL Acceptor beads to 490 μL of premixed Reaction
buffer + Activation buffer).
The Acceptor mix should be used immediately for best
results. Excess mix should be discarded.
Immediately prior to use, dilute Donor beads 20-fold in
Dilution buffer (e.g. add 10 μL Donor beads to 190 μL
Dilution buffer).
The Donor mix should be used immediately for best
results. Excess mix should be discarded.
After reconstitution in 250 μL water, lysates should be
Assay Control lysate
frozen at -20°C in single use aliquots and used within 1
month.
* Do not vortex the Reaction buffer, as vigorous mixing can damage some antibodies.
** Prepare and use Donor Mix under low-light conditions.
Control Lysate information
Control lysates are prepared from flasks of MCF7 cells (ATCC #HTB-22) at a concentration of
approximately 1 mg/mL. The controls are supplied lyophilized, and should be reconstituted in either
dd H 2 O or MilliQ® H 2 O. Once reconstituted, lysates should be stored frozen in single use aliquots.
Negative Lysate:
Prepared from MCF7 cells treated with a combination of 1 μg/mL UCN-01, 200
nM Rapamycin, and 10 μM LY294002 for 2 hours prior to lysis.
Positive Lysate:
Prepared from MCF7 cells treated with 500 ng/mL insulin for 30 minutes.
TGRKVS61.6
24 July 2015
SureFire is a registered trademark of TGR BioSciences Pty Ltd, Australia. PerkinElmer, Proxiplate, OptiPlate and AlphaScreen are registered trademarks of PerkinElmer, Inc.
page 4
4E-BP1 p-Thr37/46 AlphaScreen® SureFire® Assay Protocols
A. 2-Plate Assay - assay protocol for adherent cells
Cell Seeding
1. Seed cells (200 μL of cells for 96 well plates, 50 μL for 384 well plates) in tissue culture plates.
Incubate at 37°C overnight in serum-containing media.
Cell Treatment
2. Remove culture media, and stimulate the cells with 50 μL agonists prepared in serum-free media
(25 μL for 384-well plates). (If testing antagonists, prior to stimulation remove culture medium and
replace with 50 μL serum-free media containing antagonists (25 μL for 384-well plates)). Return cells
to 37°C incubator for desired time. 1 hour is often sufficient for signal transduction inhibitors, and 5
minutes for receptor agonists.
Note: Peptidic agonists and antagonists can often stick to plastic surfaces. To minimize this effect,
dilute in serum-free media containing a suitable carrier protein (e.g. 0.1% IgG free BSA - Jackson
Immunoresearch Cat #001-000-161).
Lysate Preparation
5. To lyse cells, remove medium from wells, and add freshly prepared 1X Lysis Buffer (50-100 μL for
a 96 well plate, 25 μL for a 384 well plate). Agitate on a plate shaker (~350 rpm) for 10 minutes at
room temperature.
6. Take 4 μL of the lysate and transfer to a 384-well Proxiplate™ for assay. (Add 4 μL Control lysates
to separate wells if required).
SureFire Assay
7. Add 5 μL of Acceptor Mix to wells. Seal plate with Topseal-A adhesive film, and incubate for 2
hours at room temperature.
8. Add 2 μL of Donor Mix to wells under subdued light. Seal plate with Topseal-A adhesive film, and
cover plate with foil. Incubate for 2 hours at room temperature.
Note: Longer incubation may give greater sensitivity. Plates can be incubated overnight if required.
9. Read plate on an Alpha Technology-compatible plate reader, using standard AlphaScreen
settings.
TGRKVS61.6
24 July 2015
SureFire is a registered trademark of TGR BioSciences Pty Ltd, Australia. PerkinElmer, Proxiplate, OptiPlate and AlphaScreen are registered trademarks of PerkinElmer, Inc.
page 5
B. 1 Plate Assay - assay protocol for non-adherent cells, and for high-throughput applications.
Note: the larger volumes required using this assay will result in achieving less assay points per kit.
Cell Seeding
1. Harvest cells by centrifugation, and re-suspend cells in HBSS at a suitable cell density. We
recommend 107 cells/mL as a starting point. Seed 4 μL of cells/well into a 384-well culture plate.
2. If using test agents/inhibitors, add 2 μL/well of 4X inhibitors prepared in HBSS.
Note: Peptidic agonists and antagonists can often stick to plastic surfaces. To minimize this effect,
dilute in serum-free media containing a suitable carrier protein (e.g. 0.1% IgG free BSA - Jackson
Immunoresearch Cat #001-000-161).
3. Return cells to incubator at 37°C for 1-2 hours.
Cell Treatment
4. Stimulate cells with agonists by addition of 2 μL/well of 4X agonist stock in HBSS containing 0.1%
BSA. The final volume in the wells should be 8 μL. (if no antagonists were used in step 2, stimulate
the cells with 4 μL/well of 2X agonist, to give a final volume in the wells of 8 μL.)
Lysate Preparation
5. To lyse the cells, add 2 μL/well 5X Lysis buffer. (Add 10 μL control lysates to separate wells if
required)
SureFire Assay
6. Add 8 μL of Acceptor Mix to wells. Seal plate with Topseal-A adhesive film, and incubate for 2
hours at room temperature.
7. Add 3 μL of Donor Mix to wells under subdued light. Seal plate with Topseal-A adhesive film, and
cover plate with foil. Incubate for 2 hours at room temperature.
Note: Longer incubation may give greater sensitivity. Plates can be incubated overnight if required.
8. Read plate on an Alpha Technology-compatible plate reader, using standard AlphaScreen
settings.
TGRKVS61.6
24 July 2015
SureFire is a registered trademark of TGR BioSciences Pty Ltd, Australia. PerkinElmer, Proxiplate, OptiPlate and AlphaScreen are registered trademarks of PerkinElmer, Inc.
page 6
Representative Data
A. Lysates were prepared at a concentration of approximately 1mg/mL from flasks of MCF7 cells
(ATCC Cat# HTB-22) treated with insulin for 60 minutes. A 2-fold dilution series of the lysate was
prepared using Lysis buffer as a diluent, and analyzed for 4E-BP1 p-Thr37/46 using the standard
2-plate protocol.
B. Lysates were prepared from plates of confluent MCF7 that had been treated with LY294002. The
lysates were analyzed for 4E-BP1 phosphorylation at either the Thr36/46 epitope, or at the
Thr70 epitope, using the standard AlphaScreen SureFire 2-plate protocol.
150000
Alpha signal (counts)
Alpha signal (counts)
200000
150000
100000
50000
0
1
10
100
1000
100000
p-Thr37/46
p-Thr70
50000
0
-10
-8
-6
-4
-2
[LY294002] M
[MCF7 lysate] %
Frequently Asked Questions & Troubleshooting
For comprehensive information on assay optimization and troubleshooting, please refer to the
following resources:
 AlphaScreen® SureFire® full manual
 Guide to AlphaScreen® SureFire® assay optimization
 AlphaScreen® SureFire® user guide
To download these resources, and other related technical information, visit
http://www.perkinelmer.com
For general information on AlphaScreen® SureFire® assays, visit http://www.tgrbio.com
TGRKVS61.6
24 July 2015
SureFire is a registered trademark of TGR BioSciences Pty Ltd, Australia. PerkinElmer, Proxiplate, OptiPlate and AlphaScreen are registered trademarks of PerkinElmer, Inc.
page 7
Customer Care
To contact the customer care team, please visit www.perkinelmer.com/ServiceCall
For more information regarding related AlphaScreen® SureFire® products and
protocols refer to:
PerkinElmer web site: www.perkinelmer.com
TGR BioSciences website: www.tgrbio.com
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
This product is not for resale or distribution except by authorized distributors.
LIMITED WARRANTY: PerkinElmer, Inc. warrants that, at the time of shipment, the products sold by it are free from defects in material and workmanship and
conform to specifications which accompany the product. PerkinElmer Inc. makes no other warranty, express or implied with respect to the products, including any
warranty of merchantability or fitness for any particular purpose. Notification of any breach of warranty must be made within 60 days of receipt unless provided in
writing by PerkinElmer Inc. No claim shall be honored if the customer fails to notify PerkinElmer Inc. within the period specified. The sole and exclusive remedy of
the customer for any liability of PerkinElmer Inc. of any kind including liability based upon warranty (express or implied whether contained herein or elsewhere),
strict liability contract or otherwise is limited to the replacement of the goods or the refunds of the invoice price of goods. PerkinElmer Inc. shall not in any case be
liable for special, incidental or consequential damages of any kind.
TGRKVS61.6
24 July 2015
SureFire is a registered trademark of TGR BioSciences Pty Ltd, Australia. PerkinElmer, Proxiplate, OptiPlate and AlphaScreen are registered trademarks of PerkinElmer, Inc.
page 8
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