(H3K27me2-1) Acceptor Beads

TECHNICAL
DATA
SHEET
AlphaLISA® Research Reagents
Caution: For Laboratory Use. A research chemical for research purposes only
Anti-di/mono-methyl-Histone H3 Lysine 27 (H3K27me2-1)
Acceptor Beads
Product No.:
AL121C (250 µg)
AL121M (5 mg)
AL121R (25 mg)
Lot No.: 1899800
Product Formats
Catalog #
Size
Volume
Assay points*
AL121C
AL121M
AL121R
250 µg
5 mg
25 mg
0.05 mL
1 mL
5 mL
500
10 000
50 000
*The number of assay points is based on an assay volume of 25 µL using a final bead concentration of 20 µg/mL in 384-well format.
Manufacturing Date:
June 3, 2014
Product Information
Description:
Anti-di/mono-methyl-Histone H3 Lysine 27 (H3K27me2-1) AlphaLISA Acceptor beads at
5 mg/mL in PBS pH 7.2, supplemented with 0.05% Proclin-300 as a preservative. Source of the
antibody: rabbit monoclonal.
Application:
This product is designed to detect human histone H3 di- and mono-methylated at lysine 27
®
(H3K27me2-1) in a homogeneous AlphaLISA assay. Broad species cross-reactivity is expected
based on sequence similarity.
Storage:
Store in the dark at 4˚C.
Stability:
This product is stable for at least 12 months from the manufacturing date when stored in its
original packaging at the recommended storage conditions.
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Quality Control
®
Lot-to-lot consistency is confirmed by a Quality Control AlphaLISA titration assay read on an EnVision Alpha HTS
instrument. Maximum signal and EC50 value are determined using the biotin-H3K27me2 peptide. Minimum signal is
derived from the non-modified biotin-H3 (21-44) peptide at the concentration giving the specified maximum signal. We
certify that this product meets our quality release criteria. Maximum counts may vary between lots and depending on
assay conditions.
Maximum signal:
Minimum signal:
EC50:
TDS-AL121-03
Page 1 of 5
980607 counts
2095 counts
16.55 nM
AlphaLISA Signal (counts)
Typical Data
600,000
biotin-H3 (21-44)
500,000
biotin-H3K27me1
biotin-H3K27me2
biotin-H3K27me3
400,000
300,000
biotin-H3K36me1
biotin-H3K36me2
biotin-H3K36me3
200,000
100,000
0
-
-10
-9
-8
-7
-6
-5
Log [biotin-peptide] (M)
Specificity of anti-di/mono-methyl-Histone H3 Lysine 27 (H3K27me2-1) Acceptor Beads. Histone H3-derived peptides with
different epigenetic marks were titrated in QC buffer (50 mM Tris-HCl pH 8.0). Signal was detected with an EnVision Alpha HTS
instrument 2102. The hook effect observed at higher peptide concentrations is typical of three-component assays and occurs when
peptide concentrations exceed the binding capacity of the Alpha Streptavidin Donor and/or AlphaLISA Acceptor beads. Shifts in affinity
might be observed in different enzymatic assay buffers.
Technical notes presenting the optimization of an AlphaLISA EZH2 histone H3 lysine 27 methyltransferase assay and
JMJD3 histone H3 lysine 27 demethylase assay using anti-H3K27me2-1 AlphaLISA Acceptor beads are available on our
website at www.perkinelmer.com/epigenetics.
Peptide Titration Assay
Peptide titration provides a means to verify product performance. It is used as our Quality Control release test. The
following reagents and materials are recommended:
Item
Supplier
Catalog #
AlphaScreen Streptavidin Donor Beads
PerkinElmer
6760002S (1 mg)
6760002 (5 mg)
6760002B (50 mg)
Histone H3K27me2 peptide, biotinylated
AnaSpec
64366
Histone H3 (21-44) peptide, biotinylated
AnaSpec
64440
AlphaLISA 5X Epigenetics Buffer 1 Kit*
PerkinElmer
AL008C (10 mL Kit)
AL008F (100 mL Kit)
White opaque OptiPlate™-384
PerkinElmer
6007290
TopSeal™-A Adhesive Sealing Film
PerkinElmer
6050195
PerkinElmer
-
®
®
®
®
EnSpire or EnVision Multilabel Plate Reader with
Alpha option
TDS-AL121-03
Page 2 of 5
*The two-component AlphaLISA 5X Epigenetics Buffer 1 Kit includes AlphaLISA 5X Epigenetics Buffer 1 and AlphaLISA 30X Buffer
Supplement. The 1X AlphaLISA Epigenetics Buffer 1 may appear cloudy. However, this will not affect assay performance. The 1X
Epigenetics Buffer 1 should be used within 16 hours.
These microplates can also be used with this product:
Item
Recommended Assay
Volume
Supplier
Catalog #
White opaque OptiPlate-96
100 µL
PerkinElmer
6005290
White ½ AreaPlate-96
50 µL
PerkinElmer
6005560
Light gray AlphaPlate™-384
25 µL
PerkinElmer
6005350
ProxiPlate™-384 Plus
12.5 µL
PerkinElmer
6008280
Light gray AlphaPlate-1536
5 - 10 µL
PerkinElmer
6004350
Recommendations

The volume indicated on each tube is guaranteed for single pipetting. Multiple pipetting of the reagents may reduce
the theoretical amount left in the tube. To minimize loss when pipetting beads, it is preferable not to prewet the tip.

AlphaScreen Donor beads are light-sensitive. All Alpha assays using the Donor beads should be performed under
subdued laboratory lighting (< 100 lux). Green filters (LEE 090 filters (preferred) or Roscolux filters #389 from Rosco)
can be applied to light fixtures.

Sodium azide should not be added to the stock reagents. High concentrations of sodium azide (> 0.001 % final in the
assay) might decrease the AlphaLISA signal.

Centrifuge the tubes briefly before use to improve recovery of content (2,000 x g, 10-15 sec). Resuspend all
reagents by vortexing before use.

Use Milli-Q grade H2O (18 MΩ•cm) to prepare the 1X AlphaLISA Epigenetics Buffer 1.

When diluting peptides, change tips after each dilution. When loading reagents in the assay microplate, change tips
after each reagent addition and between each set of reagents.

Small volumes may be prone to evaporation. It is recommended to cover microplates with a TopSeal-A Adhesive
Sealing Film to reduce evaporation during incubation. Microplates are read with the TopSeal-A Film on the plate.

Total signal varies with temperature and incubation time. For consistent results, identical incubation times and
temperature should be used for all plates.

The AlphaLISA signal is detected with an EnSpire or EnVision Multilabel Plate Reader equipped with the ALPHA
option using the AlphaScreen standard settings (i.e. Total Measurement Time: 550 ms, Excitation Time: 180 ms,
Mirror: D640as, Emission Filter: M570w, Center Wavelength 570 nm, Bandwidth 100 nm, Transmittance 75%).
®
TDS-AL121-03
Page 3 of 5
Quality Control Protocol
PLEASE READ RECOMMENDATIONS ON PAGE 3 BEFORE USE
This titration protocol is designed for 12 dilutions of 2 peptides (biotin-H3K27me2 and non-modified biotin-H3 (21-44)
peptides) with triplicate determinations. Peptide concentrations are indicated for a 10 µL volume. Final concentration of
Alpha Donor and AlphaLISA Acceptor beads in the 25 µL total assay volume is 20 µg/mL.
1)
Assay Buffer:
The Assay Buffer used for biotin-peptide dilution is 50 mM Tris-HCl pH 8.0.
2)
Serial dilutions of biotin-peptide in Assay Buffer:
Prepare dilution series for each biotin-peptide as follows, changing tip for each dilution:
Tube
Volume of
biotin-peptide
Volume of
Assay Buffer (µL)
[biotin-peptide] (M)
in 10 µL
A
B
C
D
E
F
G
H
I
J
K
L
4 µL of 500 µM stock
60 µL of tube A
60 µL of tube B
60 µL of tube C
60 µL of tube D
60 µL of tube E
60 µL of tube F
60 µL of tube G
60 µL of tube H
60 µL of tube I
60 µL of tube J
0
196
1.0E-5
3.0E-6
1.0E-6
3.0E-7
1.0E-7
3.0E-8
1.0E-8
3.0E-9
1.0E-9
3.0E-10
1.0E-10
0
140
120
140
120
140
120
140
120
140
120
100
3)
Preparation of 1X AlphaLISA Epigenetics Buffer 1:
Add 2.0 mL of AlphaLISA 5X Epigenetics Buffer 1 and 0.33 mL of AlphaLISA 30X Buffer Supplement to 7.67 mL H2O.
The cloudy appearance of the buffer is normal. Use the 1X Epigenetics Buffer 1 within 16 hours.
4)
Preparation of 5X AlphaLISA Acceptor Beads (100 µg/mL):
Add 10 µL of 5 mg/mL AlphaLISA Acceptor beads to 490 µL of 1X AlphaLISA Epigenetics Buffer 1.
5)
Preparation of 2.5X Streptavidin Donor Beads (50 µg/mL):
Keep the beads under subdued laboratory lighting.
Add 10 µL of 5 mg/mL Streptavidin Donor beads to 990 µL of 1X AlphaLISA Epigenetics Buffer 1.
TDS-AL121-03
Page 4 of 5
6)
In a white opaque OptiPlate-384 microplate:
Add 10 µL of each biotin-peptide dilution
Add 5 µL of 5X AlphaLISA Acceptor beads (20 µg/mL final)
Incubate 60 min at 23˚C
Add 10 µL of 2.5X Streptavidin Donor beads (20 µg/mL final)
Incubate 30 min at 23˚C in the dark
Read using EnSpire or EnVision Multilabel Plate
Reader with Alpha option
Please visit our website for additional information on the AlphaLISA technology at www.perkinelmer.com/AlphaTech.
This product is not for resale or distribution except by authorized distributors.
LIMITED WARRANTY: PerkinElmer BioSignal 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 BioSignal 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 otherwise provided in
writing by PerkinElmer BioSignal Inc. No claim shall be honored if the customer fails to notify PerkinElmer BioSignal Inc. within the period specified. The sole and exclusive
remedy of the customer for any liability of PerkinElmer BioSignal 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 BioSignal Inc. shall not in
any case be liable for special, incidental or consequential damages of any kind.
PerkinElmer, Inc.
940 Winter Street
Waltham, MA 02451 USA
P: (800) 762-4000 or
(+1) 203-925-4602
www.perkinelmer.com
For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs
Copyright ©2011, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners.