Biology Reference
In-Depth Information
Alternatively, if the ligand is a recombinant protein, a “tag” such as an epitope
sequence or the Fc region of IgG can be incorporated for use in the detection of
bound ligand. It should also be noted that solid-phase assays can only give
a qualitative measure of the affinity of receptor-ligand binding; other tech-
niques, such as surface plasmon resonance, must be used to calculate actual
binding parameters.
It is essential that the specificity of the assay is tested carefully. The most
important test for specificity is the ability of unlabeled ligand to compete with
labeled ligand for binding to the receptor. Hence, in the presence of a large
excess of unlabeled ligand, very little binding of labeled ligand should be
observed. Some receptor-ligand interactions are divalent-cation dependent.
Here, omitting divalent cations from the binding buffer should reduce binding
to similar levels as to wells coated with BSA alone. Further tests for specificity
can also be carried out, e.g., attempting to block the interaction using mono-
clonal antibodies to either receptor or ligand. If a specific interaction is
observed, the sequences in the receptor and ligand involved in this recognition
can be identified, using, e.g., proteolytic fragmentation, synthetic peptides, and
site-directed mutagenesis.
2. Materials
2.1. Biotinylation of Ligand
1. Coupling buffer: 0.5
M
NaCl, 0.1
M
NaHCO
3
. Dissolve 29.2 g of NaCl and 8.4 g
of NaHCO
3
in 1 L of water; the pH should be approx 8.0 without further adjustment.
2. Sulfo-NHS biotin (Pierce).
3. Tris-buffered saline (TBS). Dissolve 8.77 g of NaCl and 3.03 g of Tris in
~900 mL H
2
O, adjust pH to 7.4 using conc. HCl, and adjust final volume to 1 L
by further addition of H
2
O.
4.
TBS-azide: Add 2.5 mL of 20% (w/v) sodium azide stock solution to 1 L of TBS.
2.2. Solid-Phase Assay
1.
Dulbecco's PBS (Gibco-BRL, Gaithersburg, MD).
2.
Blocking solution: TBS, 5% (w/v) BSA, 0.05% (w/v) sodium azide. This is con-
veniently made from TBS to which sodium azide is added from a 20% stock
solution. BSA (Sigma, Madison, WI, fraction V) is added and dissolved by vig-
orous stirring. The solution is then centrifuged in 50-mL tubes (Becton-
Dickinson, Rutherford, NJ) for 5 min at 3,500
g
, and filtered through a 20-mL
disposable column (Bio-Rad, Richmond, CA). Store for <3 mo at 4
°
C.
3.
Binding buffer: TBS containing 0.1% (w/v) BSA. Divalent cations 1 m
M
MgCl
2
,
1 m
M
CaCl
2
, or 1 m
M
MnCl
2
may be added. This buffer is conveniently prepared
from TBS and stock solutions of 1
M
MgCl
2
(20.3 g MgCl
2
· 6H
2
O/100 mL), 1
M
CaCl
2
(14.7 g CaCl
2
· 2H
2
O/100 mL), or 1
M
MnCl
2
(19.8 g MnCl
2
· 4H
2
O/100 mL;
MnCl
2
is used in assays involving integrins). BSA (Sigma 99% pure grade) is
then added and dissolved by stirring. Prepare fresh.
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