Conjugation Kit

AlphaScreen® Research Reagents
Caution: For Laboratory Use. A research reagent for research purposes only
Conjugation Kit
Product No.: 6760000K
Lot No.: 1972377
Material Provided
2 x 2 mg*
* Note: 1mg translates into 2,000 assay points is based on a final bead concentration
of 20 µg/mL in a 25 µL/well reaction volume.
Manufacturing Date:
December 1, 2014
Kit Components:
Unconjugated Acceptor Beads at 20 mg/mL in
0.1M MES, pH 6.0
Streptavidin Donor Beads at 5 mg/mL in 25mM
Hepes, 0.1M NaCl, 0.05% proclin, pH 7.4
2 x 50 µL
2 x 200 µL
Product Information
This product is stable for at least 12 months from the manufacturing date if used
and stored under recommended conditions.
Storage Conditions:
Store undiluted at 4°C protected from light. Freeze-thaw is not recommended and
can cause the beads to form aggregates.
Recommended use:
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.
For additional information on running AlphaScreen assays or on potential interfering compounds, please visit our
Conjugation Protocol
Note: The following procedure is recommended only as a general guideline for bead conjugation to IgG molecules. It is highly
recommended that conditions are further optimized as required for the application for which the beads are intended.
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Aldehyde beads: (Provided)
Sodium cyanoborohydride solution: Dissolve 25 mg of sodium cyanoborohydride (Sigma #15,615-9 or equivalent)
in 1 mL Milli-Q H2O or equivalent. Note: Prepare fresh.
MES buffer (0.1 M pH 6.0): Dissolve 2.13g MES (2-[N-Morpholino]ethanesulfonic Acid) (Sigma # M-5287 or
equivalent) in 80 mL Milli-Q H2O or equivalent. Adjust the pH to 6.0 with 10 N NaOH and make up to 100 mL total
volume. Filter through a 0.2 μm filter.
1% Tween-20: Dilute 10% Tween-20 (Pierce #28320 or equivalent) 1/10 in MES buffer.
CMO solution (0.3 M pH 5.0): Dissolve 65 mg CMO (carboxymethoxylamine hemihydrochloride) (Sigma #C1,340-8
or equivalent) in 1 mL Milli-Q H2O or equivalent. Adjust the pH to 5.0 with 10 N NaOH. Note: Prepare fresh.
TRIS buffer (0.1 M pH 8.0): Dissolve 1.21g Tris (ICN #819638 or equivalent) in 80mL deionised H2O. Adjust the pH
to 8.0 with HCl and make up to 100mL total volume. Filter through a 0.2μm filter.
1. To each tube (1 mg beads) provided add:
 12.5 μL 1% Tween-20
 0.05 mg antibody (This will provide a 20:1 bead:antibody wt:wt ratio. As antibodies are available in different
concentrations depending on the supplier, 50 μL of a 1 mg/mL solution is recommended)
 10μL Sodium cyanoborohydride solution
2. Make up to a total volume of 200 μL with 0.1 M MES pH 6.0. Incubate in the dark for 48 hours at 37 C.
Add 10 μL CMO solution. Incubate in the dark at 37 C for 1 hour.
1. Add 190 μL 0.1 M Tris buffer pH 8.0.
2. Centrifuge at 13,000 g for 30 min at 4 C.
3. Remove the supernatant with a micropipette.
4. Add 1 mL 0.1 M Tris buffer pH 8.0 to pellet.
5. Vortex and sonicate*.
6. Centrifuge at 13,000 g for 30 min at 4 C.
7. Repeat steps 3 through 6.
8. Add 200 μL of an appropriate buffer** (5 mg/mL final concentration).
9. Vortex and sonicate*.
10. Store in the dark at 4 C
*Sonicate on ice at 10 pulses of 1 second (0.5 second ON/0.5 second OFF) using a Misonix XL sonicator (Model CL4) and
HS419 microtip probe.
** Either PBS or 25 mM Hepes, 100 mM NaCl, pH 7.4 is recommended.
This product is not for resale or distribution except by authorized distributors.
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