Days Of Staining Research Articles

Evaluating the clinical significance of color stability in nanohybrid composite resins: a comparative study of local and international brands

Aims: The objective of this study was to evaluate the color stability of five nanohybrid composite resins, including locally produced Turkish brands and an international brand, after exposure to staining and brushing simulations over time. Methods: Five nanohybrid composite resins; Filtek Z550 (FLT), Dolgunn (DLG), RubyComp Nano (CMP), Nova Compo C (NVC), and Parion (PRN) were tested. Specimens underwent staining and brushing simulations designed to replicate clinical oral conditions. The staining process involved immersing the specimens in a coffee solution for 8 hours per day over a 10-day period to simulate short-term staining (t1), and for 12 days to simulate long-term staining equivalent to 1 year (t2). Brushing cycles were used to replicate the mechanical wear caused by daily oral hygiene practices. Color measurements were recorded at baseline (t0), after 10 days of staining (t1), and after 1 year of simulated staining and brushing (t2). Additional analyses were conducted using Energy-dispersive X-ray spectroscopy (EDX) and Scanning Electron Microscopy (SEM) to investigate structural and surface characteristics. Statistical analysis was performed using generalized linear models and two-way robust ANOVA to assess the significance of differences in color stability, with a significance level set at p

A comparative assessment of color stability among various commercial resin composites

ObjectivesThe aim was to evaluate the color stability of six commercial restorative resin composites after being exposed to commonly consumed beverages. Repolishing impact on the stained composite was also assessed.MethodsOne-hundred and fifty disc specimens (8 mm diameter & 3 mm thickness) were prepared from Filtek™ Universal Restorative, SDR flow+, everX Flow, G-ænial A’CHORD, G-ænial Universal Flo and G-ænial Universal Injectable. To assess the color stability in five various beverages, 25 specimens from each material were randomly distributed into five groups (n = 5), according to the utilized staining solution. Group 1: distilled water, Group 2: coffee, Group 3: red wine, Group 4: energy drink, Group 5: coke. The color changes (∆E) for all materials were measured using spectrophotometer at the baseline, after 84 days of staining and after repolishing. Data was collected and analyzed using ANOVA (α = 0.05).ResultsBoth material type and staining solution had a significant effect on the color stability of specimens (p < 0.05). Compared to other beverages, the color value of the specimens submerged in coffee and wine showed the most statistically significant (p < 0.05) mean ∆E. SDR flow + in coffee and wine presented the highest ∆E when compared to other tested materials (p < 0.05). After staining of the composites, repolishing was successful in lowering the ∆E value.ConclusionsAll the beverages had an impact on the color stability of the tested resin composites, with coffee and wine demonstrating the most significant effects. The variations in color stability varied depending on the specific material utilized. Dentists should possess awareness regarding the chemical interactions that occur between different beverages and various types of resin composites. Additionally, repolishing serves as an effective technique for eliminating surface discoloration in composite restorations.

Does the composite attachment of clear aligners compromise the effectiveness of dental bleaching? An in vitro study

This study aimed to evaluate the effectiveness of dental bleaching with hydrogen peroxide 35% on the surface below the attachments. Twenty-four blocks of bovine incisors were equally divided into 2 groups. The control group comprises the enamel surface free of attachments, whereas the attachment group comprises the enamel surface with attachment. Initial staining of samples was performed with black tea for 7 days; then, an attachment was made in the attachment group with Z250 resin and new staining for another 7 days in all samples. After staining, in-office bleaching was performed in both groups, and the attachments from the attachment group were removed. Color reading was performed with a spectrophotometer at all stages: initial (baseline), after 7 days of staining, after 14 days of staining, after immediate bleaching, and after 24 hours of bleaching. The color difference was calculated, and data analysis was performed using the t test for intergroup analysis. The results showed effective bleaching in both groups; however, there was a statistically significant difference in color change between them 24 hours after bleaching. It was concluded that the presence of attachment did not impair the action of the bleaching agent on the tooth surface.

Chemical and Structural Assessment of New Dental Composites with Graphene Exposed to Staining Agents.

Among the newest trends in dental composites is the use of graphene oxide (GO) nanoparticles to assure better cohesion of the composite and superior properties. Our research used GO to enhance several hydroxyapatite (HA) nanofiller distribution and cohesion in three experimental composites CC, GS, GZ exposed to coffee and red wine staining environments. The presence of silane A-174 on the filler surface was evidenced by FT-IR spectroscopy. Experimental composites were characterized through color stability after 30 days of staining in red wine and coffee, sorption and solubility in distilled water and artificial saliva. Surface properties were measured by optical profilometry and scanning electron microscopy, respectively, and antibacterial properties wer e assessed against Staphylococcus aureus and Escherichia coli. A colour stability test revealed the best results for GS, followed by GZ, with less stability for CC. Topographical and morphological aspects revealed a synergism between GZ sample nanofiller components that conducted to the lower surface roughness, with less in the GS sample. However, surface roughness variation due to the stain was affected less than colour stability at the macroscopic level. Antibacterial testing revealed good effect against Staphylococcus aureus and a moderate effect against Escherichia coli.

Comparison of the Degree of Staining of Computer-Aided Design-Computer-Aided Manufacture (CAD-CAM) Ceramic Veneers by Green Tea, Coffee, and Coca-Cola Using a Digital Spectrophotometer.

BACKGROUND The majority of dental professionals currently recognize lithium disilicate E-max ceramic veneers as a the most widely used, conservative, and effective cosmetic materials in dentistry. This study aimed to compare the degree of surface changes - roughness (Ra), depth (Rz), and mean color changes (ΔE₀₀) - of computer-aided design-computer-aided manufactured (CAD/CAM) ceramic veneers materials of varying thicknesses caused by staining by green tea, coffee, and Coca-Cola using digital spectrophotometer. MATERIAL AND METHODS This study was conducted at King Khalid University, College of Dentistry. Lithium disilicate glass ceramic (LDGC) material was used to create 60 rectangular slices using the CAD/CAM system. The material thickness and the type of beverage were measured. The specimens were immersed in beverages according to the manufacturer's instructions. Specimen description and tomography were completed with a 3D noncontact surface metrology using interferometry. The "VITA Easy-Shade" spectrophotometer was used to measure ΔE₀₀. It was recorded after 2 weeks for different material thicknesses after immersing samples in green tea, coffee, and Coca-Cola staining materials.f RESULTS Significant changes in ceramic thickness were found in Ra and Rz of 0.07 and 1.00 mm after 14 days of staining. Coca-Cola showed a significant difference in Ra and Rz with 1.00 mm thickness measurement compared to the 0.07 mm group with ≤ of 0.05, which was considered statistically significant. Highest ΔE₀₀ were recorded among samples stained by Coca-Cola, followed by coffee, for both thicknesses. CONCLUSIONS Those findings support previous studies using spectrophotometric analysis of staining of CAD-CAM ceramic veneers that Coca-Cola followed by coffee resulted in the greatest color ΔE₀₀ change.

Time-dependent effect of intense capsule-coffee and bleaching on the color of resin-infiltrated enamel white spot lesions: an in vitro study.

Background and ObjectivesWhite spot lesions (WSLs) are a common problem that can be conservatively managed by resin infiltration. Consumption of dark beverages such as coffee causes staining of dental hard tissues, which can deteriorate the esthetic qualities of treated WSLs. The aims of this study were to investigate the effect of dark coffee heavy consumption on ICON® resin infiltrated WSLs and the influence of bleaching on them after staining.MethodsTwenty surfaces from sound human extracted third molars were used in the study. Two square-shaped buccal and lingual window areas had artificially created WSLs (received buccal resin infiltration afterward). Using VITA Easyshade, the baseline ΔE was recorded. Espresso coffee was used to immerse all surfaces for 8 days. The color coordinates according to CIE LAB were recorded for all surfaces at 2, 4, 6, and 8 days of immersion, and ΔE was calculated at each timepoint. After 8 days, in-office bleaching was applied to all surfaces according to the manufacturer’s instructions and the color. Coordinates and ΔE were recorded. For statistical analysis, an independent sample t-test was used to compare each group. A general linear mixed model (GLMM) repeated measure ANOVA was applied for statistical analysis of L*and changes due to staining over time.ResultsAnalysis of time as a main effect on the L values of surfaces was statistically highly significant (p < 0.01). The interaction of time with surfaces under investigation and type of surface (test vs. control) as a main effect were non-significant (p = 0.47 and p = 0.35, respectively). Bleaching showed a significant difference in color lightness in both test and control surfaces compared to the mean L value at 8 days of staining (p < 0.01).ConclusionCapsule-coffee consumption gradually influences the esthetic of resin infiltration on treated teeth. However, bleaching materials might restore an esthetic shade.

Color and translucency stability of CAD/CAM restorative materials

Aim: This study assessed the color and translucency stability of a polymer infiltrated ceramic network (PICN) and compared it with a resin composite (RC) and a feldspathic ceramic (FEL). Methods: Disc-shaped samples of a PICN (Vita Enamic), a feldspathic ceramic (Vitablocks Mark II), and a resin composite (Brava block) were prepared from CAD/CAM blocks. PICN and RC surfaces were finished with a sequence of polishing discs and diamond paste. FEL samples received a glaze layer. The samples were subjected to 30-min immersions in red wine twice a day for 30 days. CIEL*a*b* coordinates were assessed with a spectrophotometer at baseline and after 15 and 30 days of immersion. Color alteration (ΔE00) and translucency parameter (TP00) were calculated with CIEDE2000. Average roughness was measured before the staining procedures. Color difference and translucency data were analyzed with repeated-measures ANOVA and Tukey’s tests. Roughness was analyzed with the Kruskal-Wallis test. Results: Roughness was similar among the experimental groups. All materials had their color alteration significantly increased from 15 to 30 days of staining. PICN reached an intermediate ΔE00 between FEL and RC at 15 days. PICN revealed a color alteration as high as the composite after 30 days. No statistical difference was observed regarding translucency. Conclusion: PICN was not as color stable as the feldspathic ceramic at the end of the study. Its color alteration was comparable to the resin composite when exposed to red wine. However, the translucency of the tested materials was stable throughout the 30-day staining.

Abstract 433: Clinical validation of a Dickkopf-1 (DKK1) chromogenic in-situ hybridization (CISH) assay for manual and image-analysis assisted pathologist interpretation

Abstract Dickkopf-1 (DKK1) is a secreted modulator of Wnt signaling that is frequently overexpressed in tumors and associated with a poor prognosis. In this study we demonstrate an approach for clinically validating a RNAscope chromogenic in situ hybridization (CISH) assay for determining the level of DKK1 RNA in Gastric (G) and Gastroesophageal (GEJ) tumor tissues according to CLIA guidelines. This two-step process validated first the performance of the wet assay along with the ability of a pathologist to manually score the CISH signal according to a dot-based H-score paradigm, and second the ability of image analysis (IA) software (Flagship Biosciences) to unbiasedly and reproducibly quantify DKK1 staining in the same set of samples. The DKK1 CISH assay for manual scoring passed all pre-determined criteria of sensitivity, specificity, accuracy, and precision. 100% of the 40 evaluated G/GEJ tissues demonstrated acceptable staining in target tumor cells and absence in non-target cells. 100% of the evaluated tissues passed sensitivity with a broad dynamic range of signal expression across target cells, and negligible background staining. Reproducibility was measured by blinded pathology scoring of a serial subset of 12 cases, resulting in 11/12 (92%) with concordant DKK1 H-scores. Accuracy was assessed with a DKK1 qPCR assay on a 20-sample subset and a significant correlation with the H-score data was observed. The IA algorithm also passed all pre-determined criteria of sensitivity, specificity, accuracy, and precision. 36/40 samples (90%) passed analytical specificity with the IA algorithm correctly classifying staining on true cells belonging to the tumor. Failed samples were attributed to non-specific alkaline phosphatase activity, which in practice would be disregarded by the reviewing pathologist. 100% of samples passed the sensitivity criteria of appropriate cell identification and classification. IA precision compared H-scores across 3 days of staining, with 11/12 (92%) cases having concordant DKK1 H-scores and an ICC of 0.9009 (95% CI: 0.7117-0.9601). Digital H-scores were highly correlated to the validated manual H-scores of the 40-sample set (r = 0.72, p &amp;lt;0.0001). Taken together, these data demonstrate a clinically validated DKK1 RNAscope CISH laboratory-derived test (LDT) for manual and IA-assisted pathologist interpretation. The DKK1 RNAscope LDT is currently being applied as part of a phase 2 clinical study of DKN-01 in combination with tislelizumab to prospectively identify previously treated G/GEJ adenocarcinoma patients with elevated DKK1 tumor expression (NCT04363801). Citation Format: Charles Caldwell, Mike Kagey, Sofia Reitsma, Will Paces, Elizabeth Bueche, Vitria Adisetiyo, Roberto Gianani. Clinical validation of a Dickkopf-1 (DKK1) chromogenic in-situ hybridization (CISH) assay for manual and image-analysis assisted pathologist interpretation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 433.

Evaluation of Antioxidant Property of Amla on Bond Strength and Color Stability of Power Bleached Teeth: An In vitro Study.

Introduction:Objective: To assess the antioxidant property of 10% amla extract in reversing the compromised bond strength and to assess the antioxidant property of 10% amla extract and Elsenz on the color stability of power bleached teeth.Materials and Methods:Ninty extracted single-rooted maxillary anterior were collected and divided as follows: The labial surfaces of 30 samples were subjected to power bleaching after which the samples were divided into three groups– Group I (control), Group II (antioxidant amla), and Group III (Elsenz) with n = 10 in each which were then stained with a coffee solution for 10 mins. The color difference was recorded with a colorimeter at baseline, after bleaching, after 7, and after 15 days of staining. sixty specimens were randomly divided into six groups (n = 10) as following: Group I (immediate bonding); Group II (bleaching + immediate bonding); Group III (bleaching + antioxidant and immediate bonding); Group IV (bleaching + 1 week storage + antioxidant + bonding); Group V (bleaching + 2 week storage + antioxidant + bonding); Group VI (bleaching + 2 week storage + bonding). All the specimens were tested for shear bond strength in universal testing machine. Statistical analysis was performed using ANOVA and Scheffe's post hoc test.Results: Significantly higher staining was observed in Group II (amla) and least with Elsenz pasteThe highest mean shear bond strength was found in Group I followed by Group V. Conclusion:Elsenz showed the least staining followed by artificial saliva. 10% Amla extract neither was effective in preventing staining of power bleached enamel nor in restoring the poor bond strength of power bleached enamel.

Color stability comparisons of different type composite resins after curing and when aged in various staining solutions

Aim: To compare the color stability of two bulkfill (ormocer based or not), two flowable (high and low viscosity) and a universal nanohybrid composite resin after curing and when aged in different staining solutions. Material and Methods: Three hundred twenty discs were made from a low-flowable nanohybrid composite (GrandioSO Heavy Flow), a flowable nanohybrid composite (Grandio Flow), a bulkfill composite (GrandioSO x-tra), an ormocer-based bulkfill composite (Admira Fusion X-tra) and a universal nanohybrid composite (Grandio SO) (n=64). Specimens were subdivided into 4 subgroups, which were immersed in one of the following solutions for 3 hours a day for 30 days: distilled water, filter coffee, red wine and coke (n=16). Color evaluations were done using a dental spectrophotometer at three evaluation stages: immediately before curing, 24 hours after curing and 30 days of staining. Results: After curing; the universal nanohybrid and bulkfill composite showed significantly greater ΔE values than other type composite resins (p0.05). Bulkfill composite was the most stained resin type in all of the staining solution subgroups (p0.05). The bulkfill composite followed by universal and flowable nanohybrid composite and the ormocer based bulkfill composite showed the lowest ΔE values after coffee staining (p0.05). When effects of staining solutions are evaluated; coffee presented the highest staining effect, red wine was the second and, coke and water was the least stained solutions in application to all type of composite resins (p0.05).Conclusions: According to this study, it can be concluded that bulkfill composites showed the least resistance to staining and the ormocers were successful in color stability. In all tested materials, color changes after curing and after coffee staining were clinically perceptible. Therefore, when choosing a particular shade, such possible color changes should be taken into consideration.

In-office bleaching efficacy on stain removal from CAD/CAM and direct resin composite materials.

To evaluate the efficacy of in-office bleaching on stain removal from stained resin composite and ceramic computer-assisted design/computer-assisted manufacturing (CAD/CAM) blocks and direct resin composites. Forty disk-shaped samples were fabricated from each of nine materials: six CAD/CAM (VITABLOCS Mark II, Paradigm MZ100, Exp Vita Hybrid Ceramic, VITA ENAMIC, Exp Kerr, and LAVA Ultimate) and three direct resin composites (Filtek Supreme, Venus Diamond, and Filtek Silorane). Samples were randomly divided into five groups (n = 8), each stained with a particular staining solution. Using a calibrated spectrophotometer and a black background, L*a*b* values were assessed before and after 120 days of staining. Samples were subjected to in-office bleaching using 40% hydrogen peroxide gel for one hour. At subsequent assessment, color change (ΔE) was calculated as the difference between L*a*b* values. Both ANOVA and the Duncan test were used to identify differences between groups (α = 0.05). Bleaching resulted in significant differences in ΔE values for all materials (P < .001). Bleaching efficacy was highly influenced by material composition and staining solution. Residual color values after bleaching for ceramic and hybrid ceramics ranged from -0.49 to 2.35, within the clinically acceptable maximum of 3.3. Values after bleaching for resin-based CAD/CAM ranged from -0.7 to 7.08 while direct resin composites values ranged from -1.47 to 25.13. Coffee left the greatest residual color on all materials. Based on material nature, 40% hydrogen peroxide bleaching can remove staining. The new resin-based CAD/CAM blocks showed promising results in terms of color stability. Bleaching using 40% hydrogen peroxide can be an effective method to remove stains from dental restorations. In this way, restoration replacement as a result of discoloration may no longer be necessary.

Abstract 763: Analytical validation of Ki67/CD8 duplex IHC assay using computational tissue analysis (cTATM)

Abstract Chromogenic multiplex immunohistochemistry (IHC) assays enable investigation of the spatial relationships between tumor and immune cells, which is thought to be important for understanding and predicting therapeutic response. Development and analytical validation of multiplex IHC assays enables the use of such assays to simultaneously investigate multiple biomarkers as predictors of clinical response. In this study, we analytically validated a chromogenic duplex IHC assay that quantifies Ki67 and CD8 in formalin-fixed, paraffin-embedded non-small cell lung cancer tissues. Five performance criteria were selected and evaluated based on Clinical Laboratory Standards Institute guidelines: reportable range, analytical sensitivity, analytical specificity, accuracy, and precision. Similar to analytical validation studies for monoplex IHC assays, this study utilized a reference method and multiple days of staining. The percentage of cells positive for Ki67 nuclear staining and/or CD8 membrane staining were quantified using our computational Tissue Analysis (cTATM) platform. Performance of the Ki67/CD8 chromogenic duplex IHC assay was considered acceptable for the five criteria evaluated. Once the performance of the assay was established, additional exploratory cTA-based endpoints were examined, including the quantification of each biomarker in the tumor compartment and the tumor microenvironment, and analysis of the spatial arrangement of immune cells relative to tumor cells. In conclusion, Flagship’s cTA platform allows for more consistent quantification of individual analytes on dual-stained tissue sections, enabling investigation of complex biological questions that cannot be achieved with traditional tissue-based manual endpoints. Citation Format: Staci J. Kearney, Joshua C. Black, Benjamin J. Landis, Sally Koegler, Brooke Hirsch, Roberto Gianani. Analytical validation of Ki67/CD8 duplex IHC assay using computational tissue analysis (cTATM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 763. doi:10.1158/1538-7445.AM2017-763

Three-dimensional visualisation of the internal anatomy of the sparrowhawk (Accipiter nisus) forelimb using contrast-enhanced micro-computed tomography.

BackgroundGross dissection is a widespread method for studying animal anatomy, despite being highly destructive and time-consuming. X-ray computed tomography (CT) has been shown to be a non-destructive alternative for studying anatomical structures. However, in the past it has been limited to only being able to visualise mineralised tissues. In recent years, morphologists have started to use traditional X-ray contrast agents to allow the visualisation of soft tissue elements in the CT context. The aim of this project is to assess the ability of contrast-enhanced micro-CT (μCT) to construct a three-dimensional (3D) model of the musculoskeletal system of the bird wing and to quantify muscle geometry and any systematic changes due to shrinkage. We expect that this reconstruction can be used as an anatomical guide to the sparrowhawk wing musculature and form the basis of further biomechanical analysis of flight.MethodsA 3% iodine-buffered formalin solution with a 25-day staining period was used to visualise the wing myology of the sparrowhawk (Accipiter nisus). μCT scans of the wing were taken over the staining period until full penetration of the forelimb musculature by iodine was reached. A 3D model was reconstructed by manually segmenting out the individual elements of the avian wing using 3D visualisation software.ResultsDifferent patterns of contrast were observed over the duration of the staining treatment with the best results occurring after 25 days of staining. Staining made it possible to visualise and identify different elements of the soft tissue of the wing. Finally, a 3D reconstruction of the musculoskeletal system of the sparrowhawk wing is presented and numerical data of muscle geometry is compared to values obtained by dissection.DiscussionContrast-enhanced μCT allows the visualisation and identification of the wing myology of birds, including the smaller muscles in the hand, and provides a non-destructive way for quantifying muscle volume with an accuracy of 96.2%. By combining contrast-enhanced μCT with 3D visualisation techniques, it is possible to study the individual muscles of the forelimb in their original position and 3D design, which can be the basis of further biomechanical analysis. Because the stain can be washed out post analysis, this technique provides a means of obtaining quantitative muscle data from museum specimens non-destructively.

Comparative Effects of Turmeric, Coffee, and Chewable Tobacco on the Color Stability of Tooth-colored Restorative Materials

Objective: To compare the staining intensity of turmeric and chewable tobacco with that of coffee and to investigate the impact of staining status on the bleaching efficacy of discolored restorative materials. Study Design: A total of 72 specimens from two types of dental composites (nanocomposite, and microhybrid composite) and two types of glass ionomer cements (conventional glass ionomers, and resin-modified glass ionomers) were immersed in different staining media including turmeric, coffee, and chewable tobacco (3 Hour/day). The color changes were measured at 15th, 30th, and 45th days of staining according to the CIELAB system. The discolored specimens were then bleached using a 10% carbamide peroxide solution (2×30 min daily) and the color changes were quantified after 7 and 14 days of bleaching. The obtained data were further analysed using ANOVA with complementary Tukey's test (p<0.05). Results and Conclusion: Chewable tobacco displayed the most overall staining effect on the selected materials, followed by coffee and turmeric. Nanocomposites showed the highest vulnerability to color change due to the exposure to coffee and turmeric, while the highest color change by chewable tobacco was observed in the resin-modified glass ionomers. In contrast, the conventional glass ionomers were least stained in all staining solutions. Carbamide peroxide also exhibited more bleaching effect on nanocomposites compared to other selected restorative materials. Clinical Significance: Conventional glass ionomers and microhybrid composites are more suitable compared to resin-modified glass ionomers and nanocomposites for utilization in places where esthetic longevity is the prime concern (anterior teeth and premolars in smile line) and patient has high intake of coffee, turmeric, or chewable tobacco.

The MADS transcription factor XAL2/AGL14 modulates auxin transport during Arabidopsis root development by regulating PIN expression

Elucidating molecular links between cell-fate regulatory networks and dynamic patterning modules is a key for understanding development. Auxin is important for plant patterning, particularly in roots, where it establishes positional information for cell-fate decisions. PIN genes encode plasma membrane proteins that serve as auxin efflux transporters; mutations in members of this gene family exhibit smaller roots with altered root meristems and stem-cell patterning. Direct regulators of PIN transcription have remained elusive. Here, we establish that a MADS-box gene (XAANTAL2, XAL2/AGL14) controls auxin transport via PIN transcriptional regulation during Arabidopsis root development; mutations in this gene exhibit altered stem-cell patterning, root meristem size, and root growth. XAL2 is necessary for normal shootward and rootward auxin transport, as well as for maintaining normal auxin distribution within the root. Furthermore, this MADS-domain transcription factor upregulates PIN1 and PIN4 by direct binding to regulatory regions and it is required for PIN4-dependent auxin response. In turn, XAL2 expression is regulated by auxin levels thus establishing a positive feedback loop between auxin levels and PIN regulation that is likely to be important for robust root patterning.

RAMA stain: A fast, sensitive and less protein-modifying CBB R250 stain

SDS-PAGE and CBB staining are two of the most popular methods used for protein analysis. Although many reports that describe such staining methods have been published, these conventional protocols require several hours or days for staining and de-staining. In this study we describe a recently developed, fast and sensitive CBB staining method that utilizes the staining solution of RAMA that consists of the low-cost reagents: CBB R250, acetic acid, methanol and ammonium sulfate, and the destaining solution of water. Our method dose dependently detects 12 nanograms protein within 60 min and with a wide protein spectrum. Although the features of the dose-dependent relationship depend upon protein amounts and protein types, for most of the protein samples tested, a linear relationship was observed in the region from 12 to 330 ng. Moreover, through further washing, the detection sensitivity of protein is enhanced and reaches a maximum at 1.4 ng and then gradually decreases in the de-staining process. It has been shown recently through MS analyses that the sensitive colloidal CBB staining methods frequently result in artifactual methylations due to the strong acid and long contact during staining and the destaining processes. Such artifacts were reported to be reduced by the replacement of strong inorganic acid with acetic acid and because RAMA utilizes acetic acid and is in contact with the proteins for a short time during staining and de-staining, it is expected that in vitro artifacts will be reduced. Finally, MS analyses of RAMA-stained protein bands were revealed not to have been methylated.