Biomedical Engineering Reference
In-Depth Information
original concentration, the binding reaction is complete. This procedure takes 4 h
to overnight.
5. Remove the supernatant after the binding reaction is complete by spinning down
the beads for 5 min at 2,000 rpm.
6. Remove excess antibody by washing the beads with 5 slurry volumes of cou-
pling buffer.
7. Block remaining active groups with the ethanolamine buffer. Add a volume
equal to the original supernatant volume, and rotate overnight at 4 °C or 2 h at
room temperature.
8. Remove excess uncoupled antibody by washing the beads 4 times alternating
between coupling buffer and acetate buffer. Use a sintered glass filter, and a wash
volume at least 5 times the original slurry volume.
9. Suspend the beads in 1 slurry volume of sodium azide buffer, and store them at
4 °C.
7.3.3
Affinity Olfactory Receptor Purification Using Rho-Tag
Monoclonal Antibody
1. Rho1D4 monoclonal antibody-coupled sepharose beads
2. DPBS (Life Technologies, 14190-250)
3. DNase1 (Life Technologies
,
18047-019)
4. RNaseA (Life Technologies, 12091-039)
5. Sterile filtered water (0.22 µm) with a resistivity of at least 18 MΩ-cm.
6. Wash buffer: 0.2 % fos-choline-14 (FC14) (Anatrace/Affymatrix). This is made
from a 10 % FC14 stock solution in DPBS.
7. Elution buffer: 800 µM elution peptide Ac-TETSQVAPA-NH
2
(with an acety-
lated N-terminus and amidated C-terminus) dissolved in wash buffer.
8. High pH buffer: 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5.
9. Low pH buffer: 0.1 M sodium acetate, 0.5 M NaCl, pH 4.5.
7.3.4
Olfactory Receptor Purification Using 1D4 Rho-Tag
Monoclonal Antibody
1. Pipette the necessary amount of antibody-coupled beads into a fresh tube. Mix
the beads first by gently shaking them to ensure that they are homogeneously
suspended. The binding capacity of fresh beads is ~ 0.7 mg/ml, and the capacity
of regenerated beads is ~ 0.35 mg/ml.
2. Wash the beads with DPBS to remove excess sodium azide. Spin the beads
down at 1,400 rpm for 1 min, and then let them sit for a minute to allow them to
completely settle to the bottom of the tube. Using a pipette, slowly remove the
supernatant without disturbing the bead pellet. Add one bead volume of DPBS
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