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Protocol
Antibody Microarrays: Label plasma or serum samples
Acc.no:
2GTPKG9
|
Published:
2010-12-17
by
martin
|
Author:
Christoph Schröder
|
Affiliation:
German Cancer Research Center
Keywords: sample labeling, antibody microarray, fluorescent dye, hapten, biotin, plasma, serum, urine
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.
Instructions
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Label reaction
- 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 (see Note 10).
- Adjust volume to 200 µL with H2O.
- Per reaction, add 37.5 µL from a master mix consisting of
- 12.5 µL 20% Triton stock solution
- 25 µL 1 M sodium bicarbonate stock (pH 9.0).
- 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.
References
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Dual-color proteomic profiling of complex samples with a microarray of 810 cancer-related antibodies. ,
Author: Schröder C, Jacob A, Tonack S, Radon TP, Sill M, Zucknick M, Rüffer S, Costello E, Neoptolemos JP, Crnogorac-Jurcevic T, Bauer A, Fellenberg K, Hoheisel JD. | Date: May 2010 -
Antibody Microarrays for Expression Analysis ,
Author: Schröder C, Jacob A, Rüffer S, Fellenberg K, Hoheisel JD | Date: March 2010 -
Broken reference