Biology Reference
In-Depth Information
4.
Extravidin peroxidase reagent (Sigma).
5.
ABTS reagent: Prepare ABTS buffer: 0.05 M Na 2 HPO 4 (0.69 g Na 2 HPO 4 · 2H 2 O/
100 mL), 0.1 M sodium acetate (1.36 g CH 3 COONa · 3H 2 O/100 mL). Adjust pH
to 5.0 using conc. HCl. Store at room temperature for <3 mo. ABTS solution:
Dissolve 11 mg ABTS (Sigma) in 0.5 mL water. Prepare fresh. H 2 O 2 solution:
67
L 30% (w/w) H 2 O 2 (Sigma) mixed with 7 mL water. Prepare fresh. Add
0.5 mL of ABTS solution to 10 mL of ABTS buffer and 100
µ
L of H 2 O 2 solution.
Mix thoroughly. This amount of reagent is sufficient for one full 96-well
plate assay.
µ
6.
2% (w/v) SDS. Dissolve 2 g sodium dodecyl sulfate in 100 mL water.
3. Methods
3.1. Biotinylation
1.
Dialyze ligand into coupling buffer. About 0.5 mL of ligand at a concentration of
approx 0.5 mg/mL gives sufficient material for a large number of assays.
2.
Add an equal mass of sulfo-NHS biotin: protein (approx 0.25 mg) to the
dialysate in a 1.5-mL Eppendorf tube and mix on a rotating platform for half
hour at room temperature.
3.
To remove unincorporated biotin, dialyze the solution against two changes of 1 L
of TBS, and once against 1 L of TBS-azide.
4.
Centrifuge the dialysate in a 1.5-mL Eppendorf tube for 15 min at maximum
speed in a microcentrifuge. This removes any large aggregates or precipitate from
the solution.
5.
Store the supernatant at 4
C for <6 mo. Alternatively, many biotinylated proteins
can be stored in aliquots at -70
°
°
C.
6.
Measure the concentration of biotinylated protein using, for example, the BCA
assay (Pierce).
3.2. Solid-Phase Assay
1.
Dilute purified receptor to 1-10
µ
g/mL with Dulbecco's PBS ( see Note 1 ).
2.
Add the diluted receptor to the wells of an ELISA plate (100
L /well) ( see Note 2 ).
Leave a set of wells empty for measuring binding of the ligand to BSA. We
normally perform the assay using 4-6 replicates for each set of binding conditions.
µ
3.
Cover the plate with plastic film and store at room temperature overnight. Alter-
natively, the plate can be stored at 4
°
C for up to 1 wk.
4.
Add 25
L of blocking solution to each receptor-containing well using a multi-
channel pipet. Then remove the solution by aspiration, or by inverting the plate
over a sink and flicking out the liquid ( see Note 3 ).
µ
5.
Add 200
L of blocking solution to each well (including those used for testing
binding to BSA alone) using a multichannel pipet. Leave at room temperature for
1-3 h ( see Note 4 ).
µ
6.
Aspirate or flick out the blocking solution, and wash the wells three times with
200
µ
L of binding buffer.
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