AlphaLISA Research Reagents TruHits Kit

AlphaLISA® Research Reagents
Caution: For Laboratory Use. A research chemical for research purposes only
TruHits Kit
Product No.:
Lot No.: 1628834
Product Formats
Catalog #
Assay points*
1 000
10 000
* The number of assay points is based on an assay volume of 25 µL using a final bead concentration of 10 µg/mL in 384-well format.
Manufacturing Date:
May 15, 2012
Kit components:
AlphaLISA BSA-biotin Acceptor beads stored in
PBS, 0.05% Proclin-300, pH 7.2
1 x 50 µL at 5 mg/mL
1 x 500 µL at 5 mg/mL
Streptavidin Alpha Donor beads stored in 25 mM
Hepes, 100 mM NaCl, 0.05% Proclin-300, pH 7.4
1 x 50 µL at 5 mg/mL
1 x 500 µL at 5 mg/mL
Product Information
The AlphaLISA Acceptor beads are coated with biotinylated bovine serum albumin (BSA). The
Alpha Donor beads are coated with streptavidin.
This product is designed as a tool for AlphaLISA users to identify false positives in AlphaLISA
screening assays.
Store in the dark at 4ºC.
This product is stable for at least 12 months from the manufacturing date when stored in its
original packaging under recommended storage conditions.
Page 1 of 4
Quality Control
Lot-to-lot consistency is confirmed by a Quality Control one point 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:
821 749 counts
11 331 counts
Description of the AlphaLISA TruHits Assay
The AlphaLISA TruHits kit is designed as a tool for AlphaLISA users to identify false positives in AlphaLISA HTS assays
early in the screening process. Some compounds can interfere with the detection part of the assay, thereby artificially
reducing AlphaLISA signal.
This kit includes AlphaLISA BSA-biotin Acceptor beads and Streptavidin Alpha Donor beads which interact together to
generate an AlphaLISA signal. The excitation of the Donor beads provokes the release of singlet oxygen molecules that
triggers a cascade of energy transfer in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm. The
AlphaLISA TruHits kit allows the identification of inner filters, light scatterers (insoluble compounds), singlet oxygen
quenchers and biotin mimetics interfering with the AlphaLISA signal, and thus facilitates the detection of false positives.
However, TruHits kits do not allow systematic identification of all interfering compounds. Pan-assay interfering
substances (PAINS) such as rhodanines will not cause a decrease in TruHits AlphaLISA signal.
One Point 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:
Suggested Source
Catalog #
(1X) Phosphate-Buffered Saline (PBS), pH 7.2
White OptiPlateâ„¢-384
TopSealâ„¢-A Adhesive Sealing Film
EnSpire or EnVision Multilabel Alpha Reader
Page 2 of 4
Quality Control Protocol
This protocol provides a method to verify kit performance and is not representative of an assay. The maximum and
background signals represent an average of 6 replicates. Background signal is obtained in the presence of free biotin.
Final concentration of AlphaLISA BSA-biotin Acceptor beads and Streptavidin Alpha Donor beads is 10 µg/mL, in the
25 µL final assay volume.
Without Biotin
With Biotin
Maximum signal
Preparation of 1.7X Biotin (17 µM):
Add 17 µL of 500 µM Biotin stock solution to 483 µL of 1X PBS, pH 7.2.
Preparation of 5X Streptavidin Alpha Donor beads (50 µg/mL):
Keep the beads under subdued laboratory lighting.
Add 5 µL of 5 mg/mL Alpha Donor beads to 495 µL of 1X PBS, pH 7.2.
Preparation of 5X AlphaLISA BSA-biotin Acceptor beads (50 µg/mL):
Add 5 µL of 5 mg/mL AlphaLISA Acceptor beads to 495 µL of 1X PBS, pH 7.2.
In a microtube:
Add 300 µL of 1X PBS, pH 7.2
Add 300 µL of 1.7X Biotin (10 µM final)
Add 100 µL of 5X Alpha Donor beads (10 µg/mL final)
Add 100 µL of 5X AlphaLISA Acceptor beads (10 µg/mL final)
Incubate 30 min at 23ºC in the dark
In a white opaque OptiPlate-384 microplate:
Distribute 25 µL in 6 replicates (-/+ biotin)
Incubate 10 min at 23ºC in the dark
Read using EnSpire or EnVision Multilabel Alpha Reader
Page 3 of 4
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 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 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.
When reagents are added in the microplate, make sure the liquids are at the bottom of the well.
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.
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%).
Please visit our website for additional information on the AlphaLISA technology at
This product is not for resale or distribution except by authorized distributors.
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