Bio-Rad ProteinChip Antibody Capture Kit - Manual (Page 6)

© 2007 Bio-Rad Laboratories, Inc.
Step 1: Coupling Antibody to the ProteinChip Array
1. Thaw the TNF-
α antibody and TNF-α antigen on ice.
2. Reconstitute 750 µg of the bovine IgG provided in the kit with 100 µl PBS to
obtain a final concentration of 7.5 mg/ml.
3. Add 4 µl reconstituted control bovine IgG to 146 µl of PBS to obtain a final
concentration of 0.2 mg/ml.
4. Place a ProteinChip PG20 array on a clean, flat surface.
5. Add 2 µl of the diluted bovine IgG to spot A on the array.
6. Add 2 µl of the TNF-
α antibody to spot B on the array.
7. Add 2 µl 0.2 mg/ml antibody of interest to the remaining spots on the array.
8. Immediately transfer the array to a humidity chamber and incubate for 1 hour at
room temperature or overnight at 4°C. If larger sample volumes are being used
with a ProteinChip bioprocessor, incubate on a rocking platform.
9. Remove the bovine IgG and the TNF-
α antibody from the array using a pipet
to prevent cross-contamination during the following wash steps. Do not touch
the surface of the array with the pipet tip.
10. Place the entire array into a 15 ml conical tube containing 8 ml wash buffer
and agitate on a rocking platform for 10 minutes at room temperature.
11. Pour off the wash buffer and add 8 ml PBS to the tube and agitate on a rocking
platform for 5 minutes at room temperature.
12. Pour off the PBS and repeat step 1.11 once for a total of two washes.
13. After the PBS washes, gently blot the excess buffer from the array surface
using a lint-free lab wipe without touching the active spots on the array.
Step 2: Crosslinking Antibody to Protein G (Optional)
1. Reconstitute the crosslinking reagent as follows: Warm the crosslinking reagent
to room temperature, dissolve 0.5 mg (one vial) of crosslinking reagent by
adding 1 ml of PBS into the vial, and vigorously vortex the vial.
2. After removing the buffer from the array (step 1.13), air-dry the array for
approximately 5 minutes. Do not allow the spots to dry completely. If the
spots dry, add 1 µl PBS to each spot on the array.
3. Add 1 µl of crosslinking reagent to each spot on the array.
4. Immediately transfer the array to a humidity chamber and incubate for
30 minutes at room temperature.
5. Add 1 µl deactivation buffer to each spot, transfer the array to the humidity
chamber, and incubate with agitation for 15 minutes at room temperature.
6. Blot the excess buffer from the surface of the array using a lint-free lab wipe
without touching the active spots on the array.