Protein measurement using bicinchoninic acid: elimination of interfering substances

Brown, Rhoderick E.; Jarvis, Kari L.; Hyland, Kristi J.

doi:10.1016/0003-2697(89)90101-2

Cited by 624 publications

(322 citation statements)

References 15 publications

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“…The slurry was desalted with molecular exclusion columns packed with Sephadex G-25, diluting the sample with 100 mM Tris-HCl buffer (pH 8.0) and quantified in a molecular absorption spectrophotometer using the bicinchoninic acid method (Brown et al, 1989). Superoxide dismutase (SOD) activity was determined by measuring its capacity to inhibit the photochemical reduction of the chloride of nitrobluetretrazolium (NBT) (Giannopolitis and Ries, 1977).…”

Section: Enzyme Determinationsmentioning

confidence: 99%

Effect of salt stress on antioxidant enzymes and lipid peroxidation in leaves in two contrasting corn, Lluteño and Jubilee

Carrasco-Ríos

Pinto

2014

Chilean J. Agric. Res.

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Section: Enzyme Determinationsmentioning

confidence: 99%

Effect of salt stress on antioxidant enzymes and lipid peroxidation in leaves in two contrasting corn, Lluteño and Jubilee

Carrasco-Ríos

Pinto

2014

Chilean J. Agric. Res.

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“…After sonication and extrusion, non-encapsulated rhodamine or insulin were separated from the liposome suspension by ultracentrifugation (Himac CS 150GX, HITACHI, Tokyo, Japan) at 50,000x g, at 4°C for 4 hr or 30 min, respectively. After preparation of liposomes, the recovered amount of insulin and phospholipids were determined by BCA method as described previously (Brown et al, 1989) and Phospholipids C test-Wako (Wako Pure Chemical, Osaka, Japan), respectively. The recovery ratio of insulin and lipid were calculated as the recovered insulin and lipid divided by the applied ones, multiplied by 100.…”

Section: Preparation Of Liposomesmentioning

confidence: 99%

Noninvasive and persistent transfollicular drug delivery system using a combination of liposomes and iontophoresis

Kajimoto

Yamamoto

Watanabe

et al. 2011

International Journal of Pharmaceutics

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Iontophoresis is a promising technique for enhancing transdermal administration of charged drugs. However, conventional iontophoresis is not sufficient for effective delivery of large, hydrophilic, or electrically neutral molecules. In this study, we utilized charged liposomes as carriers, focused on a transfollicular route for delivery of the liposomes, and optimized iontophoretic conditions and lipid composition for this method in both in vitro and in vivo conditions.As a result, we identified the optimum condition (lipid composition:DOTAP/EPC/Chol=2:2:1, current supply: 0.45 mA/cm 2 , duration: 1 hr) for effective iontophoretic delivery of aqueous solution, which can not be transferred into the skin without charged liposomes. We also examined the pharmacological effects of iontophoresis of liposomes encapsulating insulin (INS-lipo) using a rat model of type I diabetes. Interestingly, iontophoresis of INS-lipo onto a diabetes rat skin resulted in a gradual decrease in blood glucose levels, with levels reaching 20% of initial values at 18 hr after administration. These lower blood glucose levels were maintained for up to 24 hr. Significant amount of insulin were also detected in plasma 18 hr after iontophoresis of INS-lipo. We succeeded in developing a non-invasive and persistent transfollicular drug delivery system that used a combination of liposomes and iontophoresis.

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“…The interference of phospholipids with the BCA assay was reported to produce a similar absorbance peak as BSA but the combined absorbance of protein and phospholipid was not strictly additive [3]. Similarly, glucose, mercaptoethanol, and dithiothreitol also produced the same absorbance response as BSA and an identical absorbance peak at 562 nm [4].…”

mentioning

confidence: 99%

“…Phospholipids [3], glucose [4], mercaptoethanol [4], and other substances such as acetamidophenol, 3,4-dihydroxyphenylalanine, dithiothreitol, glutathione, and penicillamine [2] also interfere with the BCA assay. There is also a temperature-dependent reaction of peptide bonds with Cu 2+ that can increase the concentration of Cu 1+ .…”

mentioning

confidence: 99%

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay

Vashist

Zhang

Zheng

et al. 2011

Analytical Biochemistry

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This study revealed a major interference from sulfo-N-hydroxysuccinimide (sulfo-NHS) in the bicinchoninic acid (BCA) protein assay. Sulfo-NHS, a common reagent used in bioconjugation and analytical biochemistry, exhibited absorbance signals and absorbance peaks at 562 nm, comparable to bovine serum albumin (BSA). However, the combined absorbance of sulfo-NHS and BSA was not strictly additive. The sulfo-NHS interference was suggested to be caused by the reduction of Cu 2+ in the BCA Kit's reagent B (4% cupric sulfate) in a manner similar to that of the protein.Crown Copyright Ó 2011 Published by Elsevier Inc. All rights reserved.The bicinchoninic acid (BCA) 1 protein assay [1] is widely used for determination of protein concentrations ranging from 25 to 2000 lg/mL. As a water-soluble sodium salt, BCA exhibits high sensitivity and specificity for Cu 1+ . Similar to the Biuret reaction, the presence of protein in an alkaline medium reduces Cu 2+ to Cu 1+ . Consequently, a purple-colored reaction product is formed by the chelation of two molecules of BCA with each Cu 1+ , which exhibits a strong absorbance at 562 nm. The total protein concentration is reflected by the sample color change from green to purple in proportion to a given protein concentration. The color formation caused by the reaction of protein with BCA is dependent on its number of peptide bonds, its macromolecular structure, and the availability of four amino acids (cysteine, cystine, tryptophan, and tyrosine) in the protein [2]. The amino acid sequence, pI, and the presence of certain side chains or prosthetic groups can also affect the color formation significantly. The extent of the color formation is determined by more than the sum of individual color-forming functional groups [2].The Thermo Scientific BCA protein assay kit's instruction (Cat. No. 23227) indicates several potential interfering substances such as ascorbic acid, catecholamine, creatinine, cysteine, EGTA, impure glycerol, hydrogen peroxide, hydrazides, iron, lipids, melibiose, phenol red, impure sucrose, tryptophan, tyrosine, and uric acid. Phospholipids [3], glucose [4], mercaptoethanol [4], and other substances such as acetamidophenol, 3,4-dihydroxyphenylalanine, dithiothreitol, glutathione, and penicillamine [2] also interfere with the BCA assay. There is also a temperature-dependent reaction of peptide bonds with Cu 2+ that can increase the concentration of Cu 1+ . There are numerous reports where proteins have been quantified by the BCA protein assay in the presence of sulfo-NHS [5][6][7][8][9][10][11][12][13]. However, the interference of sulfo-NHS in the BCA assay has not been reported.1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Thermo Scientific) and sulfo-NHS (Thermo Scientific)-based heterobifunctional crosslinking of proteins is one of the most widely used procedures for biomolecular conjugation [14][15][16] and biochipbased assay development [17][18][19]. Our results in this study show the interference of sulfo-NHS with the BCA protein assay, which can ...

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Protein measurement using bicinchoninic acid: elimination of interfering substances

Cited by 624 publications

References 15 publications

Effect of salt stress on antioxidant enzymes and lipid peroxidation in leaves in two contrasting corn, Lluteño and Jubilee

Effect of salt stress on antioxidant enzymes and lipid peroxidation in leaves in two contrasting corn, Lluteño and Jubilee

Noninvasive and persistent transfollicular drug delivery system using a combination of liposomes and iontophoresis

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay

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Protein measurement using bicinchoninic acid: elimination of interfering substances

Cited by 624 publications

References 15 publications

Effect of salt stress on antioxidant enzymes and lipid peroxidation in leaves in two contrasting corn, &#145;Lluteño&#146; and &#145;Jubilee

Effect of salt stress on antioxidant enzymes and lipid peroxidation in leaves in two contrasting corn, &#145;Lluteño&#146; and &#145;Jubilee

Noninvasive and persistent transfollicular drug delivery system using a combination of liposomes and iontophoresis

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay

Contact Info

Product

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About

Effect of salt stress on antioxidant enzymes and lipid peroxidation in leaves in two contrasting corn, Lluteño and Jubilee

Effect of salt stress on antioxidant enzymes and lipid peroxidation in leaves in two contrasting corn, Lluteño and Jubilee