Bio-Rad ProteinChip Antibody Capture Kit User Manual

Page 7

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.