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ProteinChip® Array

Quality Control of Antibodies

ProteinChip(R) arrays and the PBSII system can be used for an “SDS-PAGE” like analysis to:

- check purity

- analyze for presence or absence of a particular molecule

- test the ability of proteins to be detected (i.e. ionized)

Quality Control:

1. Add 1µl of protein solution to each spot on a NP20 or H4 (preferred for

antibody QC) array.The ideal protein concentration is 200-2000nM (10 fold

less for peptides)

2. Dry protein solution onto chip surface. If protein or antibody is pure and there

are no contaminating substances in the buffer such as salts or detergents

proceed to step 4.

3. If buffer contains detergents or salts, add 5µl of distilled water to the spot and

draw liquid in and out 5-10 times.

4. Air dry the chip array and add 1 ul EAM. Analyse using the SELDI ProteinChip

reader.

ProteinChip® Arrays H4 & H50

(Reverse Phase)

Direct Spotting:

1. Draw an outline for each spot using a hydrophobic pen (only for H4 chips).

2. For H4 chips pretreat the spots with 2 µl of 50% acetonitrile for 1-2 min, remove

and add 2 µl of PBS for 1-2 min.

For H50 chips pretreat the spots with 2 µl of 50% acetonitrile for 5 min, remove and repeat treatment once.

3. Add 2 µl of Binding buffer to each spot incubate for 5 min

a. PBS + 0.5M NaCl (least stringent)

b. PBS (mild stringency)

c. 10-50% ACN/PBS

*Add 0.1% TFA to the binding buffer when using H50 chips.

4. Remove BB and replace with 1-7µl of sample (diluted in BB). Do not allow the

spots to air dry during sample exchange.

5. Incubate in a humidity chamber for 30 min to 1.5 hrs at RT.

6. Wash each spot with 5µl of binding buffer three times

7. Do a final ‘quick wash’ with 5 mM Tris pH 8 or 10 mM Hepes pH 7.4, let spots

dry.

8. Add 1µl of saturated EAM solution to each spot.

9. Analyse the chip using SELDI PBSII.

Bioprocessor:

1. Draw an outline for each spot using a hydrophobic pen (only for H4 chips).

2. Pretreat the spots with 2 µl of 50% acetonitrile for 1-2 min, remove and add 2 µl of

PBS for 1-2 min.

For H50 chips pretreat the spots with 2 µl of 50% acetonitrile for 5 min, remove and repeat treatment once.

3. Put chips into bioprocessor and add 50 µl of BB to each well and incubate for 5

min shaking at RT.

4. Remove BB and add 50 – 350 µl of sample (diluted in BB). Do not allow

the spots to air dry during sample exchange

5. Incubate shaking for 30 min to 1.5 hrs at RT.

6. Wash each spot with 200 µl of binding buffer for 5 min while shaking, repeat

times.

7. Do a quick wash with low molarity buffer 5 mM Tris pH 8 or 1-10 mM Hepes

pH 7.4, dry spots.

8. Add 1ul of saturated EAM solution to each spot.

9. Analyze the chip using SELDI PBSII.

Recommended Buffers

0-60% acetonitrile ± 0.1% TFA. Isoproponal or methanol can also be used. Increasing the solvent concentration will increase the selectivity of the surface. Salts will increase hydrophobic interactions and therefore can also be included in the binding buffer (50-1000mM).

Additional Notes:

A humidity chamber can be assembled by half filling an empty tip-box with water and placing damp tissue paper in the lid. The Proteinchip® array can be placed on the tip-support in this chamber to prevent evaporation from the spots