Bio-Rad ProteinChip Antibody Capture Kit - Manual (Page 7)

© 2007 Bio-Rad Laboratories, Inc.
7. Place the entire array in a 15 ml conical tube containing 8 ml of wash buffer.
Agitate on a rocking platform for 10 minutes. Pour off the wash buffer.
8. Pour off the wash buffer and add 8 ml of PBS to the conical tube. Agitate
vigorously on a shaker for 5 minutes.
9. Repeat step 2.8 for a total of two PBS washes.
Step 3: Capturing Antigens on the ProteinChip PG20 Array
1. After completing the PBS washes, pour off the buffer. Using a lint-free lab wipe,
gently blot away any excess buffer from the surface of the array without
touching the active spots on the array.
2. Dilute 1 µl of TNF-
α antigen into 19 µl of PBS, and vortex. The final
concentration will be 50 fmol/µl.
3. Add 2 µl (100 fmol) of the diluted TNF-
α antigen to spots A and B on the array.
4. Add 2 µl of sample to the remaining spots on the array (C–H), and transfer the
array to a humidity chamber.
5. Incubate the array for 1 hour at room temperature, or incubate overnight
at 4°C.
6. Place the array in a 15 ml conical tube containing 8 ml of wash buffer. Agitate
on a rocking platform for 10 minutes. Pour off the wash buffer.
7. Add 8 ml of PBS to the conical tube. Agitate on a rocking platform for
5 minutes at room temperature.
8. Pour off the PBS, add 8 ml fresh PBS, and agitate for 5 minutes.
9. Make 500 ml of a 1 mM HEPES solution by adding 500 µl of 1 M HEPES to
500 ml deionized water.
10. Pour off the PBS and rinse the array two times with 1 mM HEPES.
11. Remove the array from the conical tube, flick off the HEPES, and allow to
air-dry for approximately 10 minutes.
Step 4: EAM Preparation and Addition
1. Add 100 µl 99.8% acetonitrile and 100 µl 1.0% TFA into the vial containing
ProteinChip SPA EAM powder to obtain a final concentration of 25 mg/ml.
2. Vortex the vial for 5 minutes to dissolve the EAM powder. Alternatively,
incubate the vial for 5 minutes in a sonicating water bath.
3. Add 0.5 µl of this saturated EAM solution to each spot on the array and air-dry.
4. Repeat step 4.3 and air-dry the array completely (approximately 10 minutes)
before reading in a ProteinChip SELDI reader.