AlphaLISA Research Reagents Anti-6xHis AlphaLISA

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DATA
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AlphaLISA® Research Reagents
Caution: For Laboratory Use. A research chemical for research purposes only
Anti-6xHis AlphaLISA Acceptor Beads
Product No.:
AL128C (250 µg)
AL128M (5 mg)
AL128R (25 mg)
Lot No.: P10211
Product Formats
Catalog #
Size
Volume
Assay Points*
AL128C
250 µg
50 µL
500
AL128M
5 mg
1 mL
10 000
AL128R
25 mg
5 mL
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:
February 28, 2011
Product Information
Description:
Anti-6xHis AlphaLISA Acceptor beads at 5 mg/mL in PBS pH 7.2 with 0.05% Proclin-300 as a
preservative. The antibody used is a mouse monoclonal antibody.
Application:
This product is intended for use in homogeneous AlphaLISA assays to capture 6xHis-tagged
targets. Tris buffer is not recommended as an assay buffer since it affects product performance
in the titration assay. Alpha Donor beads must be ordered separately.
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 under 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 HTS Alpha
instrument (see protocol below). We certify that the results meet our quality release criteria. Note: maximum counts will
vary depending on assay conditions as well as between lots. This variation has no impact on assay quality.
Maximum signal:
Minimum signal:
EC50:
TDS-AL128-01
Page 1 of 4
260 762 counts
449 counts
12.11 nM
Titration Assay (Quality Control Protocol)
This protocol provides a means to verify product performance. It is used as our Quality Control release test. The following
reagents and materials are used:
Item
Suggested Source
Glutathione Alpha Donor beads
PerkinElmer
GST, 6 His-tagged
Millipore Corp.
12-523
AlphaLISA Universal Assay Buffer, 5X
PerkinElmer
AL001C (10 mL)
AL001F (100 mL)
White OptiPlate™-384
PerkinElmer
6007290
TopSeal™-A Adhesive Sealing Film
PerkinElmer
6005185
PerkinElmer
-
®
EnSpire or EnVision Multilabel Alpha Reader
Catalog #
6765300 (1 mg)
6765301 (5 mg)
6765302 (25 mg)
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.
Alpha Donor beads are light-sensitive. All steps using the Alpha Donor beads should be performed under subdued
laboratory lighting (< 100 lux). Green filters (Roscolux filters #389 from Rosco, or the equivalent) can be applied to
light fixtures. All the other assay reagents can be used under normal light conditions.
Sodium azide should not be added to 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 water (18 MΩ•cm) to dilute the 5X AlphaLISA Universal Assay Buffer.
When diluting the probe, change tips after each dilution. When loading reagents in the assay microplate, change tips
after each reagent addition and between each set of reagents.
When reagents are added in the microplate, make sure the liquids are at the bottom of the well.
1X AlphaLISA Universal Assay Buffer contains PBS, pH 7.5, 0.1% BSA, 0.01% Proclin-300. This buffer is used in the
titration assay described below (Quality Control Protocol). Optimization of the assay buffer might be necessary in
other assay types.
Small volumes may be prone to evaporation. It is recommended to cover microplates with 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.
TDS-AL128-01
Page 2 of 4
The AlphaLISA signal is detected with an Alpha-enabled EnSpire or EnVision Multilabel Reader using the
AlphaScreen standard settings (e.g. Total Measurement Time: 550 ms, Laser 680 nm, Excitation Time: 180 ms,
Mirror: D640as, Emission Filter: M570w, Center Wavelength 570 nm, Bandwidth 100 nm, Transmittance 75%).
Quality Control Protocol
This titration protocol is designed for 12 dilutions of the probe with triplicate determinations. Final concentration of
AlphaLISA Acceptor and Alpha Donor beads in the 25 µL final assay volume is 20 µg/mL. Volume of diluted reagents
should be adjusted according to total number of assay points, plate format or assay volume.
1)
Preparation of 1X AlphaLISA Universal Assay Buffer:
Add 1 mL of 5X AlphaLISA Universal Assay Buffer to 4 mL H2O.
2)
Preparation of 1.7X probe (GST, 6 His-tagged) dilutions:
Dilute probe to a 37 µM stock solution.
Prepare dilution series in 1X AlphaLISA Universal Assay Buffer as follows, changing tip for each dilution:
Tube
Volume of Probe
Volume of 1X Buffer
(µL)
A
B
C
D
E
F
G
H
I
J
K
L
8 µL of 37 µM
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
166
140
120
140
120
140
120
140
120
140
120
140
[GST, 6 His-tagged] (M)
in 15 µL
in 25 µL
(1.7X)
Final Assay Volume
1.7E-06
1.0E-06
5.1E-07
3.0E-07
1.7E-07
1.0E-07
5.1E-08
3.0E-08
1.7E-08
1.0E-08
5.1E-09
3.0E-09
1.7E-09
1.0E-09
5.1E-10
3.0E-10
1.7E-10
1.0E-10
5.1E-11
3.0E-11
1.7E-11
1.0E-11
0
0
3)
Preparation of 5X AlphaLISA Acceptor beads (100 µg/mL):
Add 5 µL of 5 mg/mL AlphaLISA Acceptor beads to 245 µL of 1X AlphaLISA Universal Assay Buffer.
4)
Preparation of 5X Alpha Donor beads (100 µg/mL):
Keep the beads under subdued laboratory lighting.
Add 5 µL of 5 mg/mL Alpha Donor beads to 245 µL of 1X AlphaLISA Universal Assay Buffer.
TDS-AL128-01
Page 3 of 4
5)
In a white opaque OptiPlate-384 microplate:
Add 15 µL of 1.7X probe dilution
Add 5 µL of 5X AlphaLISA Acceptor beads (20 µg/mL final)
Incubate 60 min at 23ºC
Add 5 µL of 5X Alpha Donor beads (20 µg/mL final)
Incubate 30 min at 23ºC in the dark
Read using EnSpire or EnVision Multilabel Alpha Reader
AlphaLISA Signal (counts)
Typical Product Data
300,000
EC50 = 12.11 nM
200,000
100,000
0
-
-11
-10
-9
-8
-7
-6
Log [6xHis-GST] (M)
Titration assay using the Quality Control protocol. Signal was detected with an EnVision Alpha instrument 2102.
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.
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