Coomassie Brilliant Blue Method Research Articles

Visible fluorescent detection of proteins in polyacrylamide gels without staining

2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5 min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization. This study shows that placing TCE in the gel before electrophoresis can eliminate the staining step. The gel is removed from the electrophoresis apparatus and placed on a transilluminator and then the protein bands develop their fluorescence in less than 5 min. In addition to being rapid this visualization method provides detection of 0.2 μg of typical globular proteins, which for some proteins is slightly more sensitive than the standard Coomassie brilliant blue (CBB) method. Integral membrane proteins, which do not stain well with CBB, are visualized well with the TCE in-gel method. After TCE in-gel visualization the same gel can then be CBB stained, allowing for complementary detection of proteins. In addition, visualization with TCE in the gel is compatible with two-dimensional PAGE, native PAGE, Western blotting, and autoradiography.

Role of Connective Tissue Growth Factor in Mediating Hypertrophy of Human Proximal Tubular Cells Induced by Angiotensin II

Background/Aims: Cellular hypertrophy is an early important pathological feature of renal diseases such as diabetic nephropathy and remnant kidney. Recent studies have demonstrated that angiotensin II (Ang II) might be a major contributor in regulating cell hypertrophy. However, the exact mechanism is still unclear. The aim of our present work was to investigate the possible role of a newly clarified fibrogenic factor, connective tissue growth factor (CTGF), in mediating the effect of Ang II-induced tubular cell hypertrophy. Methods: The cell line HK2 was grown in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal calf serum. After the cells were rested in serum-free medium for 24 h, the dose and time response of CTGF mRNA expression to Ang II stimulation was observed by RT-PCR, and protein synthesis was observed by Western blotting. The effect of anti-CTGF on Ang II (10^–7M)-induced [³H]-leucine incorporation, total protein content (Coomassie brilliant blue G250 method) and change in cellular size [determined by a scanning electronic microscope (SEM)] was also observed. The influence of anti-CTGF antibody on the cell cycle was analyzed by using a fluorescence-activated cell sorter flow cytometer. Results: The results showed that Ang II induced expression of CTGF mRNA in a time- and dose-dependent manner (p < 0.05 and 0.01, respectively). Stimulation of cells with Ang II (10^–7M) for 48 h resulted in a 92% increase in [³H]-leucine incorporation (5,584 cpm/10⁵ cells at 0 h vs. 10,741 cpm/10⁵ cells at 48 h; p = 0.01), which was significantly abolished by treatment with anti-CTGF antibody. Ang II (10^–7M) significantly increased the total protein content in HK2 cells (control: 0.169 ± 0.011 mg/10⁵ cells vs. Ang II: 0.202 ± 0.010 mg/10⁵ cells; p = 0.03), which was markedly inhibited by cotreatment with anti-CTGF antibody. The average cellular diameter determined by SEM showed that the increase in cell size induced by Ang II could be significantly inhibited by anti-CTGF antibody (control: 11.92 ± 1.62 µm, Ang II group: 20.63 ± 3.83 µm, Ang II + anti-CTGF group: 16.43 ± 3.23 µm; p < 0.01, respectively). Furthermore, the flow cytometer study showed that Ang II arrested the cell cycle at G0–G1 phase, which was significantly reversed by treatment with anti-CTGF antibody (G0–G1 percentage: in Ang II group: 76 ± 1.8%, in Ang II + anti-CTGF group: 71 ± 1.78%; p = 0.04). Conclusion: Our data are the first to clearly demonstrate that CTGF might be an important mediator of Ang II-induced renal hypertrophy, which suggests that inhibiting the production of CTGF might be the new strategy in early prevention of renal fibrosis.

Characterization and Purification of Acid Phosphatase from Ancient Human Bone

In this research, acid phosphatase was purified and characterized from approximately 3000-year-old human bones from archeological excavations. Using anion exchange chromatography, two isoenzymes, TrACP and TsACP, were isolated from the bone. TrACP and TsACP were eluted separately, with a concentration gradient, from a CM-sepharose column. The resulting TrACP was further purified on a cellulose phosphate column. The activity was determined by using pNPP as substrate. Additionally, protein was determined by the Bradford and Coomassie Brilliant Blue method. The optimum pHs of TsACP and TrACP were 6 and 5, respectively. The optimum temperatures were 0 and 10°C, respectively. Molecular weights were measured by gel filtration chromatography. The isoenzyme purity was checked with SDS-PAGE. Finally, the effects of sodium molybdate and tartrate on isoenzyme activity were determined.

The study on the relationship between serum vascular endothelial growth factor and proteinuria in adriamycin-induced nephrotic rats.

To study the relationship between serum vascular endothelial growth factor (VEGF) and proteinuria in adriamycin-induced nephrotic rats, a rat model of adriamycin-induced nephrotitis was developed by injection of adriamycin into a tail vein in a rat. At different time points, 24-h urinary protein excretion was measured by using Coomassie brilliant blue method and the serum VEGF levels detected by using ELISA assay. The interventional effect of VEGF on this model was observed. The results showed that: (1) The adriamycin-induced nephrotic syndrome rat model was developed successfully; (2) Serum VEGF levels and proteinuria were significantly increased at 7th day after intravenous injection of adriamycin. There was a positive correlation between serum VEGF levels and 24-h urinary protein excretion (r = 0.67, P < 0.05). (3) The 24-h urinary protein excretion was significantly increased in the rats receiving administration of VEGF (P < 0.05). It was concluded that VEGF might play an important role in the pathogenesis of proteinuria in adriamycin-induced nephrotic rats.

RAYLEIGH LIGHT SCATTERING STUDY ON INTERACTION OF PROTEIN WITH BERYLLON II AND ITS APPLICATION

A new quantitative determination of proteins using 2-(8-hydroxy-3,6-dislpho-1-naphthylazo)-1,8-dihydroxynapphthalene-3,6-disulphonic acid (Beryllon II) by Rayleigh Light Scattering (RLS) has been developed.Under weak acidic condition (pH 3.80) proteins can enhance the weak RLS of Beryllon greatly and quantitatively at 380nm. This method is characterized with very high sensitivity, wide linear range of determination (0.10—32.40μg ml-1 for bovine serum albumin), rapidity, and simplicity(one step and common fluorimeter). There is little or no interference from amino acids, most of the metal ions or other coexistent substances. The method was applied to the determination of protein in human plasma and the results are very close to those of the Coomassie Brilliant Blue method assay (CBB method).

Effect of bafilomycin A1, a specific inhibitor of vacuolar (V-type) proton ATPases, on the capacitation of rabbit spermatozoa

Mammalian spermatozoa maintain precisely regulated ionic gradients that must be modified during capacitation and the acrosome reaction. In other cell types, ionic gradients are mainly regulated by the presence in plasma membranes of three metabolically different types of ATPases. The modifications induced during in vitro capacitation of rabbit spermatozoa by the specific inhibition of V-type H+-ATPases with bafilomycin A were studied. We used chlortetracycline binding to rabbit spermatozoa to monitor capacitation, and the coomassie brilliant blue method to identify acrosome-reacted sperm cells. There was a significant difference between the percentage of epididymal (66 +/- 7%) and ejaculated (43 +/- 11%) spermatozoa capacitated in vitro, after a 6-h incubation period in the presence of Ca2+ without ATPase inhibitor. The presence of bafilomycin significantly reduced these numbers (25 +/- 11 and 16+/- 8%, epididymal and ejaculated spermatozoa, respectively) and eliminated the difference. Ejaculated spermatozoa capacitated in the absence of bafilomycin showed a linear increase in the percentage of acrosome reactions induced by the addition of A23187 (12 +/- 5, 23+/- 6 and 31 +/- 5 after 15, 30 and 45 min). The presence of 0.2 micromol l-1 bafilomycin during the capacitation incubation induced a significant decrease in the acrosome reaction percentages (4 +/- 2, 8 +/- 3 and 14 +/- 4 after 15, 30 and 45 min). The addition of bafilomycin after the capacitating period had no effect upon the induction of the acrosome reaction by A23187. These results indicate that vacuolar ATPases play an important role during rabbit sperm capacitation. However, once the spermatozoa have been capacitated, V-type ATPases do not have a significant participation during the acrosome reaction.

Study on the interaction of protein with Sulfonazo III by Rayleigh light scattering technique and its application

This is the first report on the determination of proteins with Sulfonazo III by Rayleigh light scattering (RLS). At pH 4.23, the weak RLS of Sulfonazo III can be enhanced greatly by the addition of proteins, resulting in three characteristic peaks. Based on this, the reaction of Sulfonazo III and proteins was studied and a new method for quantation of proteins has been developed. This method is very sensitive (0.30–30.5 μg ml –1 for bovine serum albumen), rapid (<2 min), simple (one step), tolerance of most interfering substances, and gives a close value to that of Coomassie Brilliant Blue method in the determination of proteins in human serum. Mechanistic studies show that the RLS peaks correspond to the absorption valleys of the protein-Sulfonazo III complex.

Determination of protein by its enhancement effect on the Rayleigh light scattering of carboxyarsenazo

This is the first report on the determination of proteins based on the interaction with carboxyarsenazo (CAA) by Rayleigh light scattering (RLS). At pH 4, the weak RLS of CAA can be enhanced greatly by the addition of proteins, resulting in three characteristic peaks. Based on this, the interactions of CAA with nine kinds of proteins were studied and a new quantitative determination method for proteins has been developed. This method is very sensitive (0.10–15.3 μg ml −1 for bovine serum albumin (BSA)), rapid (<2 min), simple (one step), tolerant of most interfering substances, and gives a close value to that of the Coomassie brilliant blue (CBB) method in the determination of proteins in human serum. Thus, the CAA assay can be useful for routine analytical purposes and may overcome some of the limitations of other currently employed methods. Mechanism studies show that the three RLS peaks correspond to the absorption valleys of the CAA–protein complex.

Study on Acridine Orange dimer as a new fluorescent probe for the determination of protein

A nonfluorescent dimer of Acridine Orange is formed in situ in the presence of the anionic surfactant, sodium dodecyl sulfate (SDS). Proteins labeled with Acridine Orange dimer (AOAO) show a greatly enhanced fluorometric activity compared with that of AOAO. Based on this, a new, fast and sensitive fluorescent probe for the determination of proteins was developed. The linear range of this assay is 0.66–39.8 µg mL–1. For the detection of proteins in human serum, this method gave values close to that of the conventional Coomassie Brilliant Blue (CBB) method, but the sensitivity of the method is much superior to that of the CBB method. The detection limit for BSA was 0.08 µg mL–1.

Determination of proteins by fluorescence quenching of erythrosin B

Erythrosin B (EB) binding to proteins causes a decrease in the fluorescence maximum of EB at 550 nm. Based on this, a new, fast and simple fluorescence quenching method for the determination of proteins is developed. The linear range of this assay is 1.36–20.4 μg ml −1. The method has very few interferences, most of which can be minimized by dilution. In the detection of proteins in human serum, this method gave values close to that of the Coomassie brilliant blue method.

A Mathematical Model for the Description of the Coomassie Brilliant Blue Protein Assay

The Coomassie brilliant blue dye-binding method for protein assay has become important relatively recently. The basis of the assay method is the binding of dye to protein, with production of a dye–protein complex which absorbs light intensely at 620 nm, but the mechanism of the binding process is not well understood. In this paper, two mathematical models for the binding process are developed, one involving the binding of both protonated (green) and deprotonated (blue) forms of the dye. The second model allows only binding of the blue species to proteins. These models are tested for their ability to estimate number of dye-binding sites (n) and binding constants (Kd) from protein assay data. The models are also tested for their ability to reproduce the experimental assay curve using either known values or reasonable estimates of the equation parameters. The models are shown to be approximately equal in ability to reproduce experimental data related to the protein assay, which somewhat favors the simpler of the two models. In this paper, a method for estimatingnandKdfrom standard curve-fitting procedures is established. Hitherto, binding constants were only available from assay data taken under conditions of very large molar protein/dye ratios. The possibility of protonated forms of the dye binding to proteins was not ruled out by this study, but for many purposes the use of the simple dye-binding model, in which only the deprotonated dye species binds, is sufficient.

Effects of low and high relative molecular protein mass on four methods for total protein determination in urine

Four commonly used methods for the determination of total protein in urine were compared. These were two biuret methods using different precipitants, a Ponceau S method and a Coomassie Brilliant Blue method. The protein content of the urines was also evaluated by sodium dodecylsulphate polyacrylamide gel electrophoresis. The biuret method with ethanolic phosphotungstic acid as precipitant correlated best with the Coomassie Brilliant Blue method (r = 0.944; p less than 0.001) but less well with the Ponceau S (r = 0.895; p less than 0.001) or biuret-trichloroacetic acid (r = 0.874; p less than 0.001) methods. For urines with normal electrophoretic protein patterns, the imprecise biuret-trichloroacetic acid method (cv = 18.5%) gave the greatest number of false high results (23 in 36 urines) as assessed by electrophoresis. False low results were common in low relative molecular mass (Mr) proteinuria, especially with the biuret-tricholoroacetic acid and Ponceau S methods. High Mr proteinuria rarely caused false low results. Discrepancies between methods appear to have resulted from incomplete precipitation of low Mr protein by trichloroacetic acid.

Eight-Month Longitudinal Study of Urinary Excretion of Albumin and Tubular Proteins in Diabetic Subjects

To evaluate the importance of tubular proteinuria in diabetic nephropathy, we studied the serial changes of micro-albuminuria, microproteinuria and protein patterns by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in 38 diabetic patients over 8 months. There was a significant correlation between the amount of micro-albuminuria measured by radio-immunoassay and the amount of microproteinuria quantitated by the Coomassie brilliant blue dye binding method (r = 0.976; p less than 0.0001). Micro-albuminuria of 50 mg/day was equivalent to microproteinuria of 190 mg/day. Among the 38 diabetic patients, 26 had micro-albuminuria above 50 mg/day, while 12 had micro-albuminuria below this level. There was a significant correlation between the amount of microproteinuria and haemoglobin A1, showing that the quantity of microproteinuria was affected by metabolic control. Diabetic patients with micro-albuminuria of above 50 mg/day have a significantly higher diastolic blood pressure than those below this level. Among the diabetic patients with micro-albuminuria of less than 50 mg/day, the amount of micro-albuminuria and microproteinuria remained constant, whereas progressive increases in micro-albuminuria and microproteinuria were observed among the 12 diabetic subjects with micro-albuminuria above 50 mg/day. These support the prognostic importance of this quantity of micro-albuminuria. The protein patterns as revealed by SDS-PAGE with Coomassie blue staining show a significant loss of low-molecular-weight proteins in 7 patients, which may therefore suggest tubular damage. The loss of tubular proteins persisted over a period of 8 months in all 7 subjects, and the amount gradually increased over this period.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonlinearity in protein assays by the Coomassie blue dye-binding method

The Coomassie brilliant blue (CBB) method for protein determination takes advantage of the fact that a low-pH red form of CBB reverts to a blue form when CBB binds to protein. The increase in absorbance at 595 nm of a protein-dye mixture compared to a blank containing only the dye reagent has been used to estimate the total amount of protein present in the sample mixture. A disadvantage of this method of protein determination is that the assay plot of absorbance at 595 nm versus total protein is not linear.

Preliminary studies on the acid soluble glycoproteins in serum and their diagnostic value for acute inflammatory diseases in cattle

The concentration of acid soluble glycoproteins (ASG) in serum was determined by the Coomassie brilliant blue G-250 method as a means of assessing the amount of the acute phase protein in bovine serum. The mean (+/- SD) ASG concentration in sera from 30 normal dairy cows was 98.0 (+/- 31.5) mg/dl (range 42.2-161.0 mg/dl). The mean serum ASG concentrations in cattle suffering from enzootic bovine leukosis and enzootic bovine leukosis associated with glomerulonephritis were significantly (p less than 0.001) higher than those of normal cows. Elevated ASG concentrations were observed in cows with acute clinical mastitis. Higher (1.1-2.9 fold) ASG concentrations were observed in sera from cattle on the fifth day after surgery than in those taken at the preoperative stage. The measurement of ASG in serum could be used routinely in bovine clinical medicine as a useful indicator of inflammatory processes.

The Coomassie Brilliant Blue method underestimates drug-induced tubular proteinuria.

The sensitivity of the Coomassie Brilliant Blue (CBB) procedure differs for different proteins. To investigate the impact of this variability on clinical measurements of proteinuria, we collected urine specimens from patients with glomerular disease or drug-induced tubular nephrotoxicity and compared urinary protein concentrations as determined by the CBB method with those obtained by the biuret method. The CBB method underestimated the protein concentration predicted by the biuret method by as much as eightfold in specimens with low proportions of albumin. Determinations of protein by the CBB method in serially diluted urine specimens from patients with tubular damage also deviated from linearity by as much as twofold for protein concentrations less than 1 g/L. We conclude that CBB binding is relatively insensitive for measuring proteins specifically associated with tubular damage and hence may be inappropriate for clinical use.

Can the coomassie Brilliant Blue method for urinary protein be defended?

Can the coomassie Brilliant Blue method for urinary protein be defended?

Evaluation of an improved coomassie dye binding method for urinary protein assay

The determination of urinary proteins raises many problems since several requirements, which are difficult to reconcile, have to be satisfied. The method used must be extremely sensitive as normal urine is the biological fluid which contains the least protein and yet not be subject to interference due to urinary pigments, drugs and their metabolites. Furthermore, it should resist great variations in pH or salt content and should also be specific, measuring with equal sensitivity plasma or tissue protein but not amino acids or peptides. Most of the recent literature deals with routine methods [l] and restricts the study of the sensitivity of diverse urinary proteins to commercial albumin and globulin, without taking into account the electrophoretic and clinical classification of proteinurias [2-31. Moreover, correlation studies are often performed with other routine methods and not with a reference method, although several have been proposed [4-81. The aim of this work was to determine whether the Coomassie Brilliant Blue (CBB) method, which has been shown to be equally sensitive to various cerebrospinal fluid proteins by the addition of sodium dodecyl sulfate (SDS) to the reaction (CBB-SDS method) [9,10], could be applied to urinary proteins as well. The study comprises two successive steps. The first is the collection of a variety of well-defined urinary proteins, their purification and fractionation, and comparison of their reactivity with the CBB-SDS and biuret methods. The second step includes a prospective study of urine from healthy subjects and urine samples chosen for their weak proteinuria and high content in pigments and drugs, and comparison of the results obtained with the proposed method and an ultrafiltration biuret method based on Rice [8].

Regional differences in the neuronal cytoskeleton

Regional differences in the neuronal cytoskeleton were investigated in the giant neurons of Aplysia. Using SDS-PAGE, we have compared the proteins which comprise the cytoskeletons of cell bodies and axons. Separate populations of cell bodies and axons were collected and the proteins stained by the Coomassie brilliant blue method. Individual identified cell bodies, with long segments of their axons attached, were isolated, and the proteins were labeled with the [125I]Bolton-Hunter reagent. The proteins which are stably associated with the cytoskeleton were obtained by extracting the neuronal material in a physiological buffer containing Triton X-100. As a correlative measure to the biochemical analyses, electron microscopy was performed on the cell body and axonal fractions. Our results demonstrate that the composition and biochemical properties of the cytoskeletal proteins in the neuron cell bodies differ from those associated with axons. Specifically, the amount of neurofilament proteins, designated NF60 and NF65 , is 5 times more abundant in the axon than in the cell body. The relative amounts of actin and tubulin are comparable in these two regions of the neuron. In addition, the ratio of NF60 and NF65 is different in the cell body and axon. The cell bodies contain proportionally more NF60 than the axons. However, the physical properties of the tubulin in the cell body, as measured by relative solubility, differ from that of the axon. The substantial differences between the composition of the cytoskeleton of the cell bodies and axons of Aplysia suggests that at least two distinct cytoskeletal networks exist in these neurons, one specific for the cell body and the other specific for the axon.

Determination of proteins by using the color reaction with pyrocatechol violet-molybdenum(VI) complex.

A method for the determination of proteins (as albumin) with pyrocatechol violet (PV)-molybdenum (VI) [Mo (VI)] complex in the presence of polyvinyl alcohol was established. This method is based on the formation of the PV-Mo (VI)-protein complexes, which have absorption maxima at around 680 nm in weakly acidic media. The method could be used to determine up to ca. 300 μg/10 ml of albumin at 680 nm, and is superior to the Coomassie brilliant blue method and the pyrogallol red (PR)-Mo (VI) method in terms of sensitivity and reproducibility. Recovery of albumin added to human urine was good.