Interference of sugars in the Coomassie Blue G dye binding assay of proteins

Banik, Samudra Prosad; Pal, Swagata; Ghorai, Shakuntala; Chowdhury, Sourav; Khowala, Suman

doi:10.1016/j.ab.2008.12.006

Cited by 32 publications

(21 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Since Arg and Lys are both considered to be ''basic'' amino acids, it is perhaps not surprising that, after the gums were treated at pH 2.8, conformational changes were such that no protein was detected (i.e. no binding) (Banik et al, 2009). KLTA and GCA gums would be expected to bind the dye differently because of the different relative amounts of Arg and Lys and the total levels of protein in each gum (42 and 29 residues/1000, KLTA and GCA, respectively)).…”

Section: Estimation Of Protein ''Content'' In Gum Samples (Coomassie mentioning

confidence: 98%

“…Coomassie brilliant blue is used in detection and quantification of proteins as the dye has the ability to form complex structures in solution by electrostatic and hydrophobic interactions (Banik, Pal, Ghorai, Chowdhury, & Khowala, 2009). The ''nominal'' protein content is 3% for KLTA and 2% for GCA; however, the calculated results, obtained using BSA as a standard, suggested that the assay is unreliable in terms of the absolute levels of protein present.…”

Section: Estimation Of Protein ''Content'' In Gum Samples (Coomassie mentioning

confidence: 99%

See 1 more Smart Citation

Effects of high-hydrostatic pressure and pH treatments on the emulsification properties of gum arabic

Bell

Davis

2015

Food Chemistry

View full text Add to dashboard Cite

This study investigated the emulsification properties of the native gums and those treated at high pressure (800 MPa) both at their "natural" pH (4.49 and 4.58, respectively) and under "acidic and basic" pH (2.8 and 8.0) conditions. The emulsification behaviour of KLTA gum was found to be superior to that of the GCA gum. High pressure and pH treatment changed the emulsification properties of both gums. The acidic amino acids in gum arabic were shown to play an important role in their emulsification behaviour, and mechanisms of emulsification for the two gums were suggested to be different. The highly "branched" nature of the carbohydrate in GCA gum was also thought to be responsible for the "spreading" of droplet size distributions observed. Coomassie brilliant blue binding was used to indicate conformational changes in protein structure and Ellman's assay was used to estimate any changes in levels of free thiols present.

show abstract

Section: Estimation Of Protein ''Content'' In Gum Samples (Coomassie mentioning

confidence: 98%

Section: Estimation Of Protein ''Content'' In Gum Samples (Coomassie mentioning

confidence: 99%

Effects of high-hydrostatic pressure and pH treatments on the emulsification properties of gum arabic

Bell

Davis

2015

Food Chemistry

View full text Add to dashboard Cite

show abstract

“…The Bradford assay is more commonly used to quantify GRSP than the immunoassay, because the latter is not commercially available. However, it is recognized that other thermostable soil proteins may be extracted and that the Bradford assay is not specific to proteins and thus reacts positively to other compounds such as sugars (Rosier et al , ; Janos et al , ; Banik et al , ; Gillespie et al , ). Furthermore, some studies have indicated that humic substances co‐extracted with soil protein may interfere positively with the colorimetric Bradford assay (Halvorson & Gonzalez, ; Whiffen et al , ; Redmile‐Gordon et al , ).…”

Section: Introductionmentioning

confidence: 99%

Glomalin‐related soil protein in French temperate forest soils: interference in the Bradford assay caused by co‐extracted humic substances

Jorge-Araujo

Quiquampoix

Matumoto‐Pintro

et al. 2014

European J Soil Science

View full text Add to dashboard Cite

Thermostable soil protein, known as glomalin, is an important component of soil carbon stocks. Thought to originate from endomycorrhizal fungi, Glomales, this operationally-defined fraction of soil organic matter contains proteins of diverse origin as well as non-protein material, including humic substances. Accumulation results from the balance between production/release and subsequent degradation. Quantification of the protein is subject to uncertainty because of the co-extraction of other components that interfere with the Bradford assay. We studied 10 topsoils from French temperate forests, taken from the national forest monitoring network (Renecofor). Two fractions were extracted, easily extractable (EE) at neutral pH and total extractable (T) at pH 8. Protein was quantified with the colorimetric Bradford method, either by direct calibration using bovine serum albumin (BSA) or by extrapolation of the standard addition plot of BSA. Solubilized organic matter was characterized by using absorbance at 465 and 665 nm and by three-dimensional fluorescence excitation-emission spectroscopy. Neither soil properties nor forest cover influenced glomalin-related soil protein (GRSP) content. Direct assay gave the GRSPEE to be about 1 g kg−1 soil, and GRSPT in the range 3–10 g kg−1, accounting for about 2% of soil organic carbon and about 15% of soil nitrogen. Standard addition plots indicated a two to sixfold under-estimation of protein in total extracts, caused by negative interference with the Bradford assay. The GRSPEE was correlated significantly with both estimates of GRSPT. Under-estimation of GRSPT by direct assay was not related to the E4:E6 ratio but was correlated significantly with the intensity of absorbance at either 460 or 660 nm and with one of the fluorescence peaks. We conclude that GRSPEE is not necessarily more recent than GRSPT and that both fractions may be probes of protein content, but that absolute contents may be under-estimated because of co-extracted humic substances

show abstract

“…5B). Similarly, Banik and coworkers [17] reported interference of the Ficoll polymer in the BSA determination by the Bradford assay only when present in ion concentrations above 10 wt%. Interference is connected to the ability of the sugars to compete with proteins for binging and sequestering the Coomassie dye.…”

Section: Polymersmentioning

confidence: 86%

“…On the other hand, it was shown in several studies that the Bradford assay may suffer significant interference from some compounds that may be found in protein samples. Interferences caused by the presence of detergents [15], drugs [16], sugars [17], pharmaceutical polymers [18], and some reagents and buffers [13] have already been reported in the literature. However, poor information has been reported about the interference of specific compounds used to form ATPSs.…”

Section: Introductionmentioning

confidence: 90%

Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method

Silvério

Moreira

Milagres

et al. 2012

Analytical Biochemistry

View full text Add to dashboard Cite

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na₂SO₄, (NH₄)₂SO₄, MgSO₄, LiSO₄, Na₂HPO₄, sodium phosphate buffer (pH 7.0), NaH₂PO₄, K₂HPO₄, potassium phosphate buffer (pH 7.0), KH₂PO₄, C₆H₈O₇, Na₃C₆H₅O₇, KCHO₂, NaCHO₂, NaCO₃, NaHCO₃, C₂H₄O₂, sodium acetate buffer (pH 4.5), and NaC₂H₃O₂] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5 wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10 wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents.

show abstract

Interference of sugars in the Coomassie Blue G dye binding assay of proteins

Cited by 32 publications

References 9 publications

Effects of high-hydrostatic pressure and pH treatments on the emulsification properties of gum arabic

Effects of high-hydrostatic pressure and pH treatments on the emulsification properties of gum arabic

Glomalin‐related soil protein in French temperate forest soils: interference in the Bradford assay caused by co‐extracted humic substances

Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method

Contact Info

Product

Resources

About