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
Because this drug is administered via intravitreal injection and removed after staining, significant systemic absorption is not expected. The dye is removed after clinical surgery. As the drug is administered via intravitreal injection, it may only be distributed to a specific area of the eyeball.

[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 an ophthalmic solution used to stain the internal limiting membrane (ILM) during ophthalmic surgery. This membrane is thin and translucent, making it difficult to identify in ophthalmic procedures requiring high visual precision. As its name suggests, Brilliant Blue G imparts a vibrant blue color to the ILM, facilitating its identification. It was approved by the U.S. Food and Drug Administration (FDA) for ophthalmic use on December 20, 2019.

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

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Mechanism of Action
The internal limiting membrane (ILM) is a thin, translucent structure that defines the transition zone between the retina and vitreous humor. The ILM acts like a scaffold, allowing excessive tissue to grow on it. When this tissue projects onto the adjacent retina, it causes visual distortion. This can lead to vision loss and/or visual deformities. The epiretinal membrane (also known as the ERM) is a fibrous tissue located on the inner surface of the retina. Its occurrence is of unknown cause, but it is sometimes associated with retinal detachment and inflammation. It is typically located on the surface of the internal limiting membrane (ILM), causing decreased vision and distorted vision. These conditions, along with associated macular folds or tractional macular degeneration, can affect the ILM, leading to visual complications. Removal of the ILM, with or without vitrectomy, is usually a simple treatment for these conditions. Brilliant Blue G specifically stains the intraocular ILM without staining the epiretinal membrane or the retina itself, making surgical removal easier. Currently, its specific staining mechanism limited to the internal limiting membrane (ILM) is not fully elucidated.

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Pharmacodynamics
Brilliant Blue G can assist ophthalmic surgery, making the internal limiting membrane (ILM) easier to identify and thus facilitating surgical removal.

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Coomassie Brilliant Blue (CBB) is a commonly used dye for the staining of proteins separated by SDS-PAGE, offering advantages such as simple staining operation and high quantitative accuracy. Furthermore, it is fully compatible with mass spectrometry protein identification. However, despite these advantages, CBB's sensitivity is lower than silver staining or fluorescent staining, and therefore it is rarely used for protein detection in gel-based proteomics methods. To improve the sensitivity of CBB, several improvements have been made to the original Coomassie Brilliant Blue staining protocol (1). To improve the detection sensitivity of low-abundance proteins, researchers proposed two main improvements, namely converting dye molecules into colloidal particles: In 1988, Neuhoff and colleagues added 20% methanol and a higher concentration of ammonium sulfate to the Coomassie Brilliant Blue G-250-based staining solution (2); in 2004, Candiano et al. established the blue-silver staining method based on Coomassie Brilliant Blue G-250 using phosphoric acid, ammonium sulfate, and methanol (3). However, all these improved protocols could only detect about 10 ng of protein. In 2002, Kang et al. published a little-known colloidal Coomassie Brilliant Blue staining protocol, which improved Neuhoff's colloidal Coomassie Brilliant Blue staining protocol and mainly involved complexing substances. They replaced ammonium sulfate with aluminum sulfate and replaced methanol with less toxic ethanol (4). The novel aluminum-based staining method proposed by Kang et al. exhibits excellent sensitivity, capable of detecting phosphorylase b down to 1 ng/band, and its sensitivity is less affected by the type of protein. This article will demonstrate the application of Kang et al.'s method in rapid and sensitive colloidal Coomassie brilliant blue staining for protein analysis. We will use our group's routine two-dimensional gel electrophoresis as an example to illustrate this rapid and simple method. [1] In the Bradford protein quantification method, the protein concentration is determined by the absorbance at 595 nm after Coomassie brilliant blue G-250 (CBBG) binds to the protein. In the protein-CBBG mixture, surface-enhanced Raman scattering (SERS) is very sensitive to the molecular weight of free CBBG adsorbed on the silver surface, and the amount of bound CBBG is directly related to the concentration of the target protein. Therefore, based on the SERS of free CBBG and with silicon as an internal standard, we developed a new method for detecting the total protein concentration in solution. Compared with the traditional Bradford protein assay, the proposed protein assay has two significant advantages: a wider linear concentration range (10⁻⁵-10⁻⁹ g/mL) and a 200-fold reduction in the detection limit (1 ng/mL), which suggests its great potential for rapid and highly sensitive determination of high-abundance and low-abundance protein concentrations. [2]

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Pulmonary fibrosis (PF) is a life-threatening disease with a very poor prognosis. Meeting this unmet medical need is extremely challenging due to the complexity of the pathogenesis of PF. Many lung diseases are associated with the activation of NF-κB and NLRP3 inflammasomes. Coomassie Brilliant Blue G-250 (CBBG) has been shown to be a safe and highly selective P2×7R antagonist with the potential to inhibit NLRP3 inflammasome activity. This study investigated for the first time the effect of CBBG on bleomycin-induced pulmonary fibrosis in rats. Our results showed that CBBG significantly improved the histological features and oxidative state biomarkers of bleomycin-exposed lung tissue. In addition, the analysis of hydroxyproline, TGF-β, PDGF-BB, TIMP-1, MMP-9, Col1a1, SMA and ICAM-1 showed that CBBG inhibited collagen deposition. The determination of TNF-α, IL-1β, IL-18 and MCP-1 in lung tissue also showed that CBBG has anti-inflammatory effects. The total protein and LDH activity in bronchoalveolar lavage fluid were significantly reduced. The lung protective effect of CBBG may be attributed to the inhibition of NLRP3 inflammasome on the one hand, and to the inactivation of NF-κB on the other hand. The decrease in phosphorylated p65 and its DNA binding activity and the analysis of TLR4 confirmed the inactivation of NF-κB. Inhibition of NLRP3 inflammasome assembly leads to the inhibition of Caspase-1 activity. In conclusion, CBBG can be used as a first-line or adjuvant therapy for PF, and therefore may provide a new way to meet unmet medical needs. [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.)