Antibody Microarrays: Label plasma or serum samples


Expected outcome


Label reaction of complex plasma samples for antibody microarray analysis


Abstract


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.

Reagents

  • 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

Equipment

  • Use Zeba 2 mL Desalt columns (Pierce)

Instructions

  1. 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).
  2. Thaw protein samples on ice; the protein concentration should be at least 5 mg/mL.
  3. Transfer 1 mg of each protein sample to a 1.5 mL reaction tube
  4. Adjust volume to 200 µL with H2O.
  5. Dissolve label reagent (NHS ester of hapten or fluorescent dye) in H2O to have a concentration of 8 mM.
  6. Mix thoroughly by pipetting.
  7. Use label reagents immediately after dissolving them in order to prevent hydrolysis of the NHS-esters.
  8. Per reaction, add 12.5 µL of dissolved label reagent.
  9. Incubate reaction tubes for one hour on a shaker (200 rpm) at 4°C, protected from light.
  10. To stop the reaction, add 17 µL of 50% hydroxylamine to each reaction tube and incubate for 30 min at 4°C.
  11. 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.
  12. Add 30 µL of 25 x protease inhibitor cocktail stock solution.
  13. Prepare 30 µL aliquots and store protected from light at -20°C.

Citation:
Schröder C, Jacob A, Rüffer S, Fellenberg K, Hoheisel JD., Antibody Microarrays for Expression Analysis. Antibody Engineering. March 2010, edition 2, volume 2, chapter 33, p429-445. Springer Verlag.