Biology Reference
In-Depth Information
7.
Aspirate or flick out the final wash. Remove residual liquid by inverting the plate
and striking it hard several times onto adsorbent paper toweling.
8.
Dilute the biotin-labeled ligand in binding buffer. Add 100
µ
L of this solution to
each experimental well (
see
Note 5
).
9.
Cover the plate with plastic film and incubate at 37
°
C for 3 h. A cell-culture
incubator is suitable for this purpose.
10.
Aspirate the wells to remove unbound ligand.
11.
Wash the wells three times with 200
µ
L of binding buffer. Remove residual buffer
as in
step 7
.
12.
Dilute Extravidin-peroxidase reagent 1:500 in binding buffer. Add 100 mL to
each well using a repeating pipet. Incubate the plate for 10-15 min at room tem-
perature. During this time prepare the ABTS reagent.
13.
Aspirate the wells to remove unbound Extravidin-peroxidase.
14.
Wash the wells twice with 200
µ
L of binding buffer and then twice with 400
µ
L
of binding buffer. Remove residual buffer as in
step 7
.
15.
L of ABTS reagent to each well using a repeating pipet. Allow the
reaction to proceed until a strong (but not dark) green color is obtained (typically
10-30 min).
Add 100
µ
16.
Stop the reaction by adding 100
µ
L of 2% SDS solution to each well using a
repeating pipet.
17.
Read the plate using an automatic plate reader at 405 nm.
18.
Calculate mean and standard deviations of ligand binding to experimental wells.
Subtract the level of binding to wells coated with BSA only.
4. Notes
1.
In initial experiments, the concentrations of both receptor and ligand should be
varied so that the conditions for optimal signal to background binding can be
determined. Detailed protocols for the purification of integrin receptors have pre-
viously been described
(2
,
6)
. For receptors containing detergents (e.g., Triton
X-100), it is necessary to dilute the solution so that the detergent concentration is
< 0.002% (w/v), otherwise the detergent interferes with adsorption of the recep-
tor to the plate. Plates can be coated with receptor several days in advance.
2.
Immulon 1B or 4HBX ELISA plates (Dynatech, Chantilly, VA) are suitable. More
recently, we have found that half area EIA/RIA plates (Costar, Cambridge, MA)
also work well, and have the advantage that similar results can be obtained with
half as much receptor (i.e., 50
µ
L/well).
3.
A small amount of blocking solution is added to the wells before aspirating the
receptor solution because we have found that this renders the wells hydrophilic
and prevents them drying out when they are aspirated. Drying out of the wells
may destroy the activity of some of the receptor. For the aspiration we use a 21-gage
hypodermic needle attached by tubing to a Buchner flask, which is connected to
a water pump or vacuum line.
4.
Longer blocking times (e.g., overnight) or alternative blocking reagents may be
used if the background level of binding is high.
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