Folin-Ciocalteau reagent

A method that has been the standard of choice for many years is the Lowry procedure. This method uses Cn ions along with Folin-Ciocalteau reagent, a combination of phosphomolybdic and phosphotnngstic acid complexes that react with Cn. Cn is generated from Cn by readily oxidizable protein components, such as cysteine or the phenols and indoles of tyrosine and tryptophan. Although the precise chemistry of the Lowry method remains uncertain, the Cn reaction with the Folin reagent gives intensely colored products measurable spectrophotometrically. 

Recently, a reagent that reacts more efficiently with Cn than Folin-Ciocalteau reagent has been developed for protein assays. Bieinehoninie aeid (BCA) forms a purple complex with Cu in alkaline solution. 

Lowry method uses a combination of the Biuret copper-based reagent and the Folin-Ciocalteau reagent, which contains phosphomolybdic-phosphotungstic acid. Reagents react with protein, yielding a blue colour that displays an absorbance maximum at 750 nm... 

A standard Lowry-based protein assay has been adjusted to the special conditions encountered with skin [126], Basically, proteins reduce an alkaline solution of Cu(II)-tartrate to Cu(I) in a concentration-dependent manner. Then, the formation of a blue complex between Folin-Ciocalteau reagent (a solution of complex polymeric ions formed from phosphomolybdic and phosphotungstic heteropoly acids) and Cu(I) can be measured spectrophotometrically at 750 nm. A calibration curve can be obtained by dissolving known amounts of stratum corneum in 1 M sodium hydroxide. A piece of tape that has not been in contact with skin is subjected to an identical procedure and serves as negative control. The method was recently adapted to a 96-well plate format, notably reducing analysis times [129],... 

As phenolics are well-known antioxidant agents, the Folin-Ciocalteau total phenolics assay was performed to assess phenolic content in ginseng roots. This assay was performed as described by Singleton et al. [20] with modifications [21]. Briefly, extract dissolved in 70% methanol was combined with Folin-Ciocalteau reagent, incubated for 5 min, and then 7.5% NaHCOs was added. Samples were transferred into microplate wells in triplicate, incubated in darkness for 2 h, and then absorbance read at 725 nm. Samples were blanked against a treatment with only solvent. A standard curve of quercetin was produced and data were expressed as quercetin equivalents. 

Phenol and phenolic derivatives, cresol and related compounds were analyzed by Folin-Ciocalteau reagent at 700 m/i, standardizing each compound separately by its Folin response. Their slopes (molar absorption coefficients) were 8000-10,000 M-1cm-1. Interestingly, methoxyl aromatics with no free hydroxyl groups were not reactive. 

Sadler, N.P. and Jacobs, H. 1995. Application of the folin-ciocalteau reagent to the determination of salbutamol in pharmaceutical preparations. Talanta 42 1385-1388. 

Singleton, V.L., Orthofer, R., and Lamuela-Raven-tos, R.M. 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteau reagent. Methods En-zymol. 299 152-178. 

Incubate the tubes at room temperature for 10 min. Add (and immediately mix in) 0.3 ml of Folin-Ciocalteau reagent to each of the 10 tubes. 

N Folin-Ciocalteau reagent 2% Na2C03 in 0.1 N NaOH 1% CuS04 5H20 2% sodium tartrate... 

Colour Tests. Ammoniacal Silver Nitrate (room temperature)— black Ferric Chloride—green Folin-Ciocalteau Reagent— blue Mandelin s Test—orange Marquis Test—brown Meth-anolic Potassium Hydroxide—blue - orange Nessler s... 

E 15 l2 in methanol F Folin-Ciocalteau Reagent G Madelin Reagent H Cinnamaldehyde... 

Cu ions from the Biuret reaction (see Section 1.2.1) form an unstable blue complex with the Folin-Ciocalteau reagent. This complex serves as a measure of the protein concentration. 

Disadvantages The Lowry assay requires a lot of pipetting work and many buffer components can interfere, including mercaptoethanole, HEPES, TRITON-X-100, and other detergents (they precipitate). Hence, it is advisable to precipitate the protein from the samples (see Section 1.5) and to perform the Lowry assay on the protein pellet. You have to make the reagents yourself, except for the Folin-Ciocalteau reagent available from (Merck). The Lowry protein values of different labs are difficult to compare, because every lab carries out the assay a little differently. 

Add 0.2 ml of the diluted Folin-Ciocalteau reagent to each sample and vortex immediately. 

A. Qualitative Spray with 20% sodium carbonate solution followed by Folin-Ciocalteau reagent. Gray-blue zones result. Detection limit < 0.1 Pi... 

B. Quantitative Developed with solvent A. Plates dried in warm air stream and immediately sprayed with 20% aq. sodium carbonate, dried thoroughly and sprayed with fresh Folin-Ciocalteau reagent diluted 1 3 and dried IS min in warm air stream. Scan with integrating densitometer (Photovolt model 520 M with TLC stage used slit 0.1 X 6 mm 420-nm filter search unit, 1 mm response setting, 10). Read each spot against standard curve... 

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