Label reaction of complex plasma samples for antibody microarray analysis
This protocol describes the procedure for a direct labeling of proteins in complex plasma, serum or urine samples. Samples can be either labeled directly by a fluorescent dye, or by a hapten such as biotin using NHS-ester chemistry.
- 12.5 µL 20% Triton stock solution per reaction
- 25 µL 1 M sodium bicarbonate stock (pH 9.0) per reaction
- NHS ester of hapten or fluorescent dye
- 30 µL of 25 x protease inhibitor cocktail
- Use Zeba 2 mL Desalt columns (Pierce)
- Create 37.5 µL master mix per reaction by mixing 12.5 µL 20% Triton stock solution with 25 µL 1 M sodium bicarbonate stock (pH 9.0).
- Thaw protein samples on ice; the protein concentration should be at least 5 mg/mL.
- Transfer 1 mg of each protein sample to a 1.5 mL reaction tube
- Adjust volume to 200 µL with H2O.
- Dissolve label reagent (NHS ester of hapten or fluorescent dye) in H2O to have a concentration of 8 mM.
- Mix thoroughly by pipetting.
- Use label reagents immediately after dissolving them in order to prevent hydrolysis of the NHS-esters.
- Per reaction, add 12.5 µL of dissolved label reagent.
- Incubate reaction tubes for one hour on a shaker (200 rpm) at 4°C, protected from light.
- To stop the reaction, add 17 µL of 50% hydroxylamine to each reaction tube and incubate for 30 min at 4°C.
- Use Zeba 2 mL Desalt columns (Pierce) in order to remove unreacted dye and exchange buffer to PBS according to the protocol provided by the manufacturer.
- Add 30 µL of 25 x protease inhibitor cocktail stock solution.
- Prepare 30 µL aliquots and store protected from light at -20°C.