Skip to Content
Merck
  • Protein carbonyl determination by a rhodamine B hydrazide-based fluorometric assay.

Protein carbonyl determination by a rhodamine B hydrazide-based fluorometric assay.

Redox biology (2018-05-05)
Christos D Georgiou, Dimitrios Zisimopoulos, Vasiliki Argyropoulou, Electra Kalaitzopoulou, Panayiotis V Ioannou, George Salachas, Tilman Grune
ABSTRACT

A new fluorometric assay is presented for the ultrasensitive quantification of total protein carbonyls, and is based on their specific reaction with rhodamine B hydrazide (RBH), and the production of a protein carbonyl-RBH hydrazone the fluorescence of which (at ex/em 560/585 nm) is greatly enhanced by guanidine-HCl. Compared to the fluorescein-5-thiosemicarbazide (FTC)-based fluorometric assay, the RBH assay uses a 24-fold shorter reaction incubation time (1 h) and at least 1000-fold lower protein quantity (2.5 µg), and produces very reliable data that were verified by extensive standardization experiments. The protein carbonyl group detection sensitivity limit of the RBH assay, based on its standard curve, can be as low as 0.4 pmol, and even lower. Counting the very low protein limit of the RBH assay, its cumulative and functional sensitivity is 8500- and 800-fold higher than the corresponding ones for the FTC assay. Neither heme proteins hemoglobin and cytochrome c nor DNA interfere with the RBH assay.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hydrazine hydrate, reagent grade, N2H4 50-60 %
Sigma-Aldrich
2,4-Dinitrophenol, moistened with water, ≥98.0%
Sigma-Aldrich
Hemoglobin equine, lyophilized powder
Sigma-Aldrich
Rhodamine B, ≥95% (HPLC)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Pepsin from porcine gastric mucosa, lyophilized powder, ≥3,200 units/mg protein
Sigma-Aldrich
Methanol, suitable for HPLC, ≥99.9%
Sigma-Aldrich
Sodium borohydride, ReagentPlus®, 99%
Sigma-Aldrich
21-Hydroxyprogesterone, ≥97% (HPLC)
Sigma-Aldrich
Butylated hydroxyanisole, ≥98.5%
Sigma-Aldrich
Citric acid monohydrate, ACS reagent, ≥99.0%
Sigma-Aldrich
Guanidine hydrochloride, ≥99% (titration), organic base and chaeotropic agent
Sigma-Aldrich
Deoxyribonucleic acid sodium salt from salmon testes
Sigma-Aldrich
Urea, ReagentPlus®, ≥99.5%, pellets
Sigma-Aldrich
Lysozyme from chicken egg white, powder or granules, ≥90 %, ≥39,000 units/mg protein
Sigma-Aldrich
Sodium deoxycholate, ≥97% (titration)
Sigma-Aldrich
Cytochrome c from equine heart, ≥95% (SDS-PAGE)
Sigma-Aldrich
Ammonium iron(II) sulfate hexahydrate, ACS reagent, 99%
Millipore
EDTA, Disodium Salt, Dihydrate, Molecular Biology Grade