Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions

• Derivatizing protein carbonyls with dinitrophenylhydrazine is very common.
• For the measurement of protein carbonyls in plasma, cells, organ homogenates and isolated proteins are a wide variety of sample preparation and handling protocolls used.
• ELISA seems best-suited for high-throughput clinical studies.
• Immunoblotting is especially applicable in cell culture studies.
• Existing protocols must be further improved and standardized.

Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples.

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