Dye reagent

Staining Proteins After Polyacrylamide Gel Electrophoresis Using Brilliant Blue G-250
1. Preparation of Solutions
Coomassie Staining Solution:
1) Dissolve 100 g of aluminum sulfate (14-18 hydrate) in 2000 mL of Milli-Q water.
2) Add 200 mL of 96% ethanol and mix thoroughly.
3) Add 0.4 g of Brilliant Blue G-250 and stir until fully dissolved.
4) Slowly add 47 mL of 85% phosphoric acid while stirring continuously.
5) Adjust the final volume to 2000 mL using Milli-Q water.
Note: Do not filter the solution. It should remain in a colloidal state with suspended particles.
Destaining Solution:
Take 2000 mL of Milli-Q water, add 200 mL of 96% ethanol, and 47 mL of 85% phosphoric acid.
2. Staining Procedure
Gel Handling:
1) After protein electrophoresis, carefully remove the gel from the glass plate.
2) Rinse the gel three times with Milli-Q water, 10 minutes each, to remove SDS.
3) Shake the Coomassie staining solution before use to ensure even dispersion of colloidal particles.
4) Immerse the gel in the staining solution and agitate on a shaker for 2-12 hours.
Staining Process:
Protein bands will begin to appear after 10 minutes, with 80% staining achieved within 2 hours.
For optimal staining, overnight staining is recommended.
Destaining:
1) After staining, remove the staining solution and rinse the gel twice with Milli-Q water.
2) Place the gel in the destaining solution and agitate for 10-60 minutes to remove excess dye.
3. Rinsing and Storage
1) Rinse the gel twice more with Milli-Q water to restore its original thickness.
2) The gel can be stored in the refrigerator, and adding an acidic solution can prevent mold contamination.
4. Notes
1) Ensure the gel is thoroughly washed before staining, as residual SDS may interfere with dye-protein binding.
2) The staining solution can be reused as long as particles remain in the solution.
3) It is recommended to store the staining solution in a dark bottle to extend its shelf life.

Effect of Coomassie brilliant blue G-250 (CBBG) on lung histological picture and the percentage of fibrosis area (A%) [3]
As shown in Fig. 1, lung specimens from Normal (Fig. 1A) or CBBG 1000 (Fig. 1B) animals displayed no histological alterations of normal bronchioles, alveoli, alveolar walls and airspaces; however, BLMCN-exposed rats exhibited massive inflammatory-cell infiltration, alveolar wall thickening, fibrinous exudates and the highest of both inflammation score (Fig. 1C). In contrast, CBBG at both the lowest dose (Fig. 1D) and particularly the highest dose (Fig. 1E) resulted in a marked improvement in lung histological features of lower degree of inflammatory-cell infiltration, alveolar wall thickening, and fibrinous exudates. Additionally, these groups showed a significant decline in the inflammation score (Fig. 1F) compared with that of untreated BLMCN-exposed lungs. With regards to the percentage of fibrosis area (Fig. 2), Normal (Fig. 2A) or CBBG 1000 (Fig. 2B) animals displayed no fibrotic changes. In contrast, BLMCN-exposed lungs showed increased fibrotic tissue deposition (Fig. 2C). Treatment with CBBG (500 mg/kg) (Fig. 2D) and, particularly CBBG (1000 mg/kg) (Fig. 2E) resulted in a significant decrease in the A% of fibrosis compared with that of BLMCN-treated rats (Fig. 2F).

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Effect of Coomassie brilliant blue G-250 (CBBG) on the total protein and LDH in the BALF and caspase-1 activity in the lung tissue [3]
Levels of LDH (Fig. 3a) and total protein (Fig. 3b) in the BALF were significantly increased in BLMCN-exposed rats compared with those of the Normal rats. In contrast, these levels were significantly decreased in rats treated with CBBG (500 mg/kg) and CBBG (1000 mg/kg) compared with those of the BLMCN-treated rats. Additionally, levels of LDH and total protein in the BALF were significantly decreased in rats treated with CBBG (1000 mg/kg) compared to those treated with CBBG (500 mg/kg). Further, treatment of BLMCN-exposed rats with CBBG (1000 mg/kg) resulted in a significant decrease in the levels of both LDH and total protein in the BALF compared with those of BLMCN + CBBG 500 group of rats. Similarly, both the lowest and the highest dose of CBBG resulted in a significant decrease in caspase-1 activity (Fig. 3c) and that the BLMCN-exposed rats treated with CBBG (1000 mg/kg) showed a significant decrease in that activity compared with that of the BLMCN + CBBG 500 group of rats.

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Effect of Coomassie brilliant blue G-250 (CBBG) on MDA, GSH, SOD, CAT and NOx [3]
Intratracheal BLMCN treatment resulted in a significant increase in the levels of MDA (Fig. 4a) and NOx (Fig. 4e) compared with those of the Normal rats. However, treatment with CBBG at both the lowest and the highest doses resulted in a significant decrease in the levels of MDA and NOx compared with those of the BLMCN-treated rats. On the other hand, BLMCN exposure resulted in a significant decrease in the levels of GSH (Fig. 4b), SOD (Fig. 4c) and CAT (Fig. 4d) compared with those of the Normal rats. In contrast treatment with CBBG (500 mg/kg) resulted in a significant increase in the levels of GSH and SOD and a non-significant difference in the level of CAT compared with those of the BLMCN-treated rats. Additionally, treatment with CBBG (1000 mg/kg) resulted in a significant increase in the levels of GSH, SOD and CAT compared with those of the BLMCN-treated rats. Further, regarding the levels of MDA, CAT and NOx, we detected a significant difference between BLMCN + CBBG 500 and BLMCN + CBBG 1000 groups.

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Effect of Coomassie brilliant blue G-250 (CBBG) on the mRNA expression of Col1a1 and α-SMA and the hydroxyproline content in the lung tissue [3]
As shown in Fig. 5, BLMCN treatment resulted in a significant increase in the levels of Col1a1 mRNA (Fig. 5b), α-SMA mRNA (Fig. 5c) and hydroxyproline content of lung tissue (Fig. 5a) compared with those of the Normal rats. However, treatment with CBBG (500 mg/kg) resulted in a significant decrease in the levels of both Col1a1 mRNA expression and hydroxyproline content and a non-significant change in α-SMA mRNA expression level compared with those of the BLMCN-treated rats. On the other hand, treatment with CBBG (1000 mg/kg) resulted in a significant decrease in the levels of Col1a1 mRNA, α-SMA mRNA and hydroxyproline compared with those of the BLMCN-treated rats. Furthermore, levels of COl1a1 mRNA, α-SMA mRNA and hydroxyproline were significantly decreased in rats treated with CBBG (1000 mg/kg) compared to those treated with CBBG (500 mg/kg).

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Effect of Coomassie brilliant blue G-250 (CBBG) on TNF-α, IL-1β, IL-18, TGF-β, TGF-β mRNA, PDGF-BB, MMP-9, TIMP-1, ICAM-1, MCP-1 in lung tissue [3]
With regards to the levels of TNF-α (Fig. 6a), IL-1β (Fig. 6b), IL-18 (Fig. 6c), TGF-β (Fig. 6d), TGF-β mRNA (Fig. 6e), PDGF-BB (Fig. 6f), MMP-9 (Fig. 6g), TIMP-1 (Fig. 6h), ICAM-1 (Fig. 6i), and MCP-1 (Fig. 6j), BLMCN exposure resulted in a significant increase in their levels compared with those of the Normal rats. In contrast, treatment with CBBG (500 and 1000 mg/kg) resulted in a significant decrease in their levels compared with those of the BLMCN-treated rats. Further, levels of TNF-α, TGF-β, TGF-β mRNA, PDGF-BB, MMP-9, TIMP-1, ICAM-1, and MCP-1 were significantly decreased in rats treated with CBBG (1000 mg/kg) compared to those treated with CBBG (500 mg/kg).

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Effect of Coomassie brilliant blue G-250 (CBBG) on TLR4, TLR4 mRNA, p-p-65, p65 binding activity, NLRP3, NLRP3 mRNA [3]
With regards to the levels of TLR4 (Fig. 7a), TLR4 mRNA (Fig. 7b), p-p-65 (Fig. 7c), p65 binding activity (Fig. 7d), NLRP3 (Fig. 7e), and NLRP3 mRNA (Fig. 7f), BLMCN exposure resulted in a significant increase in their levels compared with those of the Normal rats. In contrast, treatment with CBBG (500 mg/kg) resulted in a significant decrease in the levels of TLR4, TLR4 mRNA, p65 binding activity, NLRP3, NLRP3 mRNA and a non-significant change in the levels of p-p-65 compared with those of the BLMCN-treated rats. Additionally, treatment with CBBG (1000 mg/kg) resulted in a significant decrease in the levels of TLR4, TLR4 mRNA, p-p-65, p65 binding activity, NLRP3, NLRP3 compared with those of the BLMCN-treated rats. Further, levels of TLR4, TLR4 mRNA, p-p-65, p65 binding activity, NLRP3 mRNA were significantly decreased and levels of NLRP3 were insignificantly changed in rats treated with CBBG (1000 mg/kg) compared to those treated with CBBG (500 mg/kg). However, a significant change between the latter two groups could be detected upon omitting an outlier that was detected upon measuring the level of NLRP3 in PF rats treated with CBBG (500 mg/kg).

Pretreatment of Protein Samples [2]
The protein assay reagent was diluted 100 times with PBS, and then the protein samples were added, respectively, to it. After 2 min, silver colloid was added and the volume ratio of the diluted protein assay reagent, protein sample, and silver colloid was 1:1:1 and the concentration of Ag colloid used in SERS measurements is 3 × 10−4 M.

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SERS Measurement [2]
One minute after mixing the protein−CBBG mixture with the silver colloid, an amount of 10 μL of each sample was dripped onto a silicon chip for a SERS measurement. SERS spectra were measured with a HoloSpec f/1.8i spectrograph, and the 785 nm of a NIR diode laser was used as an excitation source. The laser power at the sample was about 15 mW, and the exposure time for each SERS measurement was 15 s. All SERS spectra shown here were collected with baseline correction.

Rats were randomly allocated into five experimental groups as follows: Normal group (n = 8), in which rats were subjected to the anesthetization protocol, the surgical procedure, and the intratracheal instillation of PBS; Coomassie brilliant blue G-250 (CBBG) 1000 group (n = 8), in which rats were administered Coomassie brilliant blue G-250 (CBBG) (1000 mg/kg/day; p.o.) for 4 weeks and were subjected to the anesthetization protocol, the surgical procedure, and the intratracheal instillation of PBS. This rat group served as the drug control group; BLMCN group (n = 10), in which rats were subjected to the anesthetization protocol, the surgical procedure, and the intratracheal instillation of BLMCN (5 mg/kg in PBS) for once at the first day of experiment; BLMCN + CBBG 500 group (n = 8), in which rats were administered CBBG (500 mg/kg/day; p.o.) for 4 weeks and were subjected to the anesthetization protocol, the surgical procedure, and the intratracheal instillation of BLMCN (5 mg/kg in PBS) for once at the first day of experiment; BLMCN + CBBG 1000 group (n = 8), in which rats were administered CBBG (1000 mg/kg/day; p.o.) for 4 weeks and were subjected to the anesthetization protocol, the surgical procedure, and the intratracheal instillation of BLMCN (5 mg/kg in PBS) for once at the first day of experiment. CBBG treatment were started 2 days before induction of pulmonary fibrosis. At the end of experimental protocol, rats were euthanized under secobarbital (500 mg/kg; i.p.) as an anesthetic (Table 1). CBBG doses were chosen based on previous studies in which authors reported data about drug safety and estimation of oral bioavailability. [3]

Absorption, Distribution and Excretion
Due to the fact that this drug is administered by intravitreal injection and subsequently removed after staining, it is not expected to be significantly absorbed systemically.
This dye is removed in a clinical setting after the surgical procedure in which it was used.
This drug is administered via intravitreal injection and likely distributes only to parts of the eye.

[1]. Dyballa N, Metzger S. Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels. J Vis Exp. 2009;(30):1431. Published 2009 Aug 3.

[2]. Highly sensitive protein concentration assay over a wide range via surface-enhanced Raman scattering of Coomassie brilliant blue. Anal Chem. 2010;82(11):4325-4328.

[3]. Coomassie brilliant blue G-250 dye attenuates bleomycin-induced lung fibrosis by regulating the NF-κB and NLRP3 crosstalk: A novel approach for filling an unmet medical need [published online ahead of print, 2022 Feb 21]. Biomed Pharmacother. 2022;148:112723.

Brilliant Blue G is used in an ophthalmic solution for staining the internal limiting membrane (ILM) of the eye during ophthalmic procedures. This membrane is thin and translucent, making it difficult to identify during eye surgeries that require high levels of visual accuracy. Brilliant blue G, like its name, imparts a vibrant blue color, facilitating identification of the ILM. It was approved by the FDA for ophthalmic use on December 20, 2019.

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Drug Indication
This drug is indicated for selectively staining the internal limiting membrane (ILM).
FDA Label

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Mechanism of Action
The internal limiting membrane (ILM) is a thin and translucent structure that demarcates the transition from the retina from the vitreous body of the eye. It acts as a scaffold on which excessive tissue can grow, which results in visual distortion when it is projected onto the neighbouring retina. This causes visual loss and/or distortion. An epiretinal membrane (also known as ERM) is a fibrous type of tissue that can be found on the inner surface of the retina and occurs idiopathically, and in some cases, retinal detachment and inflammation. It is often found on the surface of the internal limiting membrane (ILM), causing visual loss and distortion. The above condition as well as the associated macular pucker or traction maculopathy can affect the ILM, contributing to visual complications. The removal of the ILM with or without vitrectomy is often a simple solution to these conditions. Brilliant Blue G specifically stains the internal limiting membrane (ILM) in the eye without staining the epiretinal membrane or the retina, allowing for easier surgical removal. The mechanism of its specific staining that is limited to the ILM is currently not fully understood.

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Pharmacodynamics
Brilliant Blue G aids in ophthalmologic surgery by rendering it easier to identify the internal limiting membrane (ILM) for surgical removal.

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Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches. Several improvements of the original Coomassie protocol(1) have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution(2), and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol(3). Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol(4). The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins. Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.[1]

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In the Bradford protein assay, protein concentrations are determined by the absorbance at 595 nm due to the binding of Coomassie brilliant blue G-250 (CBBG) to proteins. In a protein-CBBG liquid mixture, surface-enhanced Raman scattering (SERS) is sensitive to the amount of unbound CBBG molecules adsorbed on silver surfaces, and the bound CBBG amount is directly related to the target protein concentration. Accordingly, a novel method for detecting total protein concentration in a solution has been developed based on SERS of unbound CBBG with an internal standard of silicon. Two obvious advantages of the proposed protein assay over conventional Bradford protein assay are its much wider linear concentration range (10(-5)-10(-9) g/mL) and 200 times lower limit of detection (1 ng/mL), which demonstrates its great potential in rapid, highly sensitive concentration determination of high and low-abundance proteins.[2]

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Pulmonary fibrosis (PF) is a life-threatening disorder with a very poor prognosis. Because of the complexity of PF pathological mechanisms, filling such an unmet medical need is challenging. A number of pulmonary diseases have been linked to the activation of NF-κB and the NLRP3 inflammasome. Coomassie brilliant blue G-250 (CBBG) is proved to be a safe highly selective P2×7R antagonist with promising consequent inactivation of NLRP3 inflammasome. This is the first report to investigate the effect of CBBG on the bleomycin-induced lung fibrosis in rats. Our findings revealed that CBBG resulted in a significant improvement in histological features and oxidative status biomarkers of bleomycin-exposed lung tissue. Additionally, CBBG repressed collagen deposition as indicated after the analysis of hydroxyproline, TGF-β, PDGF-BB, TIMP-1, MMP-9, Col1a1, SMA and ICAM-1. It also exhibited anti-inflammatory potential as revealed by the determination of TNF-α, IL-1β, IL-18, MCP-1 in the lung tissue. In the bronchoalveolar lavage, the total protein and the LDH activity were substantially reduced. The lung protective effects of CBBG might be attributed on the one hand to the inhibition of NLRP3 inflammasome and on the other hand to the inactivation of NF-κB. Decreased levels of phospho-p65 and its DNA-binding activity as well as the analysis of TLR4 confirmed NF-κB inactivation. Caspase-1 activity is suppressed as a consequence of inhibiting NLRP3 inflammasome assembly. To conclude, CBBG may act as a primary or adjuvant therapy for the management of PF and therefore it may pose an opportunity for a novel approach to an unmet medical need.[3]

C47H48N3NAO7S2

854.02

853.283

C, 66.10; H, 5.67; N, 4.92; Na, 2.69; O, 13.11; S, 7.51

6104-58-1

6324599

Dark purple to black solid powder

>1.0 g/cm3 (20ºC)

100 °C

11 °C

11.198

1

9

13

60

1610

0

CCN(CC1=CC(=CC=C1)S(=O)(=O)[O-])C2=CC(=C(C=C2)/C(=C\3/C=CC(=[N+](CC)CC4=CC(=CC=C4)S(=O)(=O)[O-])C=C3C)/C5=CC=C(C=C5)NC6=CC=C(C=C6)OCC)C.[Na+]

RWVGQQGBQSJDQV-UHFFFAOYSA-M

InChI=1S/C47H49N3O7S2.Na/c1-6-49(31-35-11-9-13-43(29-35)58(51,52)53)40-21-25-45(33(4)27-40)47(37-15-17-38(18-16-37)48-39-19-23-42(24-20-39)57-8-3)46-26-22-41(28-34(46)5)50(7-2)32-36-12-10-14-44(30-36)59(54,55)56;/h9-30H,6-8,31-32H2,1-5H3,(H2,51,52,53,54,55,56);/q;+1/p-1

sodium;3-[[4-[(Z)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-N-ethyl-3-methylanilino]methyl]benzenesulfonate

Acid blue 90; Brilliant Blue G; 6104-58-1; C.I. Acid Blue 90; Coomassie Brilliant Blue G; Acid Blue 90; C.I. Acid Blue 90, monosodium salt; M1ZRX790SI; Coomassie Blue G 250; Coomassie Brilliant Blue G; BBG; Brilliant Blue G

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~12.5 mg/mL (~14.64 mM)
H2O : ~10 mg/mL (~11.71 mM)

Solubility in Formulation 1: ≥ 0.56 mg/mL (0.66 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.6 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.56 mg/mL (0.66 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.6 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)