Furthermore, it also increases the insulin secretion. Federal government websites often end in .gov or .mil. [3] Under acidic conditions, the red form of the dye is converted into its blue form, binding to the protein being assayed. PMC For the "G" variant the blue colour has a more greenish tint. It is an organic dye that makes complexes with basic amino acids, such as lysine, histidine, tyrosine, and arginine. Figure 6. [12] The entire experiment is done at room temperature. When SDS concentrations are below critical micelle concentration (known as CMC, 0.00333%W/V to 0.0667%) in a Coomassie dye solution, the detergent tends to bind strongly with the protein, inhibiting the protein binding sites for the dye reagent. We found that the neutral ionic species of CBB binds to proteins by a combination of hydrophobic interactions and heteropolar bonding with basic amino acids. The dye molecules bind to proteins, including those in wool (keratin), to form a proteindye complex. The equation displayed on the chart gives a means for calculating the absorbance and therefore concentration of the unknown samples. ScienceDirect.com | Science, health and medical journals, full text . Under the acid conditions the dye is normally a brownish colour but on binding to the protein the blue form of the dye is produced. PDF Coomassie Blue (R-250, G-250) - INTERCHIM Stain the mini-gel with enough Invitrogen SimplyBlue SafeStain (20-100 ml) to cover the gel. In the red form, all three nitrogen atoms carry a positive charge. They soaked the gel in a dye solution containing methanol, acetic acid and water. 2023;2611:285-291. doi: 10.1007/978-1-0716-2899-7_15. Halmkov., J. The only reported side effect was that the rats temporarily turned blue. please go to the Copyright Clearance Center request page. Coomassi Blue Staining | Thermo Fisher Scientific - US We would know nothing about the microorganisms around us if this incredible instrument did, Need Pipettes for your Lab? Coomassie Brilliant blue R 250 (C.I. 42660) - MilliporeSigma Material Safety Data Sheet or SDS for Coomassie Brilliant blue G 250 (C.I. Materials supplied by user: You will need following items for Coomassie Blue Staining. 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. The Coomassie Brilliant Blue G-250 dye has three forms: anionic (blue), neutral (green), and cationic (red). Federal government websites often end in .gov or .mil. When the dye binds to the proteins through a process that takes about 2 minutes, a change in the absorption maximum of the dye from 465nm to 595nm in acidic solutions occurs. and transmitted securely. With this procedure it was no longer necessary to destain the gel. This method is also time sensitive. Repeat Steps 9-11 for two times, or shake the gel in reagent D for 15 minutes at room temperature. The pKa values for the losses of the two protons are 1.15 and 1.82, respectively. Clipboard, Search History, and several other advanced features are temporarily unavailable. The study presented that the female samples have a higher absorbance as compared to the male samples when tested at similar wavelengths. 2013 Mar 15;434(2):287-91. doi: 10.1016/j.ab.2012.11.014. The animal experiments administered the dye within 15 minutes of injury, but to be effective in a real-life setting, where it may take time for a patient to reach the emergency room, the treatment needs to be effective even when administered up to two hours after injury. [1] During the formation of this complex, the red form of Coomassie dye first donates its free electron to the ionizable groups on the protein, which causes a disruption of the protein's native state, consequently exposing its hydrophobic pockets. Testing on the rats proved effective. Mazowieckie | province, Poland | Britannica Although these modifications result in a less sensitive assay, a modified method becomes sensitive to detergents that can interfere with sample.[20]. The Coomassie stain can interact with a small group of amino acids: arginine, histidine, lysine, phenylalanine, tyrosine, and tryptophan making it a useful stain for fingerprint analysis to identify the biological sex of the fingerprint originator. To prevent hazardous, flammable vapors from forming, do not allow the solution to boil. Clipboard, Search History, and several other advanced features are temporarily unavailable. Our studies show Coomassie Brilliant Blue G-250 as a promising chemical chaperone that stabilises the -helical native human insulin conformers, disrupting its aggregation. If there's no protein to bind, then the solution will remain brown. 2014 Elsevier Inc. All rights reserved. Keywords: [9], The procedure for Bradford protein assay is very easy and simple to follow. Wash the mini-gel with 100 ml of water for 1-3 hours. Language links are at the top of the page across from the title. Ideally, the R2 value will be as close to 1 as possible. Thermo Fisher Scientific. Coomassie Brilliant Blue - an overview | ScienceDirect Topics DISCLAIMER: ConductScience and affiliate products are NOT designed for human consumption, testing, or clinical utilization. Place one or two stained gels in a staining container containing the 100 ml destain solution. Abstract This protocol describes Coomassie brilliant blue staining, one of the most common methods of detecting proteins in polyacrylamide gels (PAGE). Coomassie R-250 and G-250 dyes are two chemical forms of a disulfonated triphenylmethane compound that is commonly used as the basis of stains for detection of proteins in gel electrophoresis and Bradford-type assay reagents for protein quantitation. Disclaimer. Scatchard analysis showed that the number of Coomassie R ligands bound to each protein molecule is approximately proportional to the number of positive charges on the protein, about 1.5-3 dye molecules/charge. [7], Many protein-containing solutions have the highest absorption at 280nm in the spectrophotometer, the UV range. Coomassie Brilliant Blue R-250 | C45H45N3NaO7S2+ | CID 23693030 - structure, chemical names, physical and chemical properties, classification, patents, literature . MeSH The following elaborates on how one goes from the standard curve to the concentration of the unknown. An official website of the United States government. Compute the extinction coefficient and calculate the concentrations of the unknown samples. After electrophoresis follow the instructions below. Caution: Use caution while performing the following steps using a microwave oven. Life (Basel). Can be stored at room temperature. To obtain the clearest background for photography, perform a second 1 hour wash with 100 ml water. 2015;1312:41-7. doi: 10.1007/978-1-4939-2694-7_7. Le., C. Gently shake the gel at room temperature on an orbital shaker until the desired background is achieved. 2010 Sep 15;404(2):193-6. doi: 10.1016/j.ab.2010.05.022. Wojewdztwo mazowieckie - Wikimedia Commons Coomassie Brilliant Blue R-250 | C45H45N3NaO7S2+ | CID 23693030 - PubChem km and is Poland's largest province in terms of both area and population. The dye also forms a complex with the anionic detergent sodium dodecylsulfate (SDS). This protocol describes Coomassie brilliant blue staining, one of the most common methods of detecting proteins in polyacrylamide gels (PAGE). Mazowieckie Province - Poland Coomassie blue staining; G-250; Kimwipes or paper towels; Polyacrylamide gels (PAGE); R-250; Silver staining; Whatman 1 filter paper. Stain overnight or longer if needed. Place a small piece of laboratory tissue in the solution to absorb excess dye. From Wikimedia Commons, the free media repository. Loosely cover the staining container and heat in a microwave oven at full power for 1 minute. If you have a specific question about products available in your area, please contact your local sales office or representative. These pockets in the protein's tertiary structure bind non-covalently to the non-polar region of the dye via the first bond interaction (van der Waals forces) which position the positive amine groups in proximity with the negative charge of the dye. Protein samples were separated electrophoretically on a cellulose acetate sheet. By clicking Submit, you acknowledge that you may be contacted by Fisher Scientific in regards to the feedback you have provided in this form. The binding of the dye to a protein causes a shift in the absorbance maximum of the dye from 465 to 595nm. It is an organic dye that makes complexes with basic amino acids, such as lysine, histidine, tyrosine, and arginine. Sodium dodecyl sulfate (SDS), a common detergent, may be found in protein extracts because it is used to lyse cells by disrupting the membrane lipid bilayer and to denature proteins for SDS-PAGE. Coomassie brilliant blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. 2. Coomassie Brilliant Blue G-250 Dye: An Application for Forensic Fingerprint Analysis. See also: Ready-to-use Coomassie Brilliant Blue R-250 staining and destaining solutions (161-0435) Discard each rinse. The "G" form of the dye has been studied in detail. The sheet was then soaked in sulfosalicylic acid to fix the protein bands and transferred to a solution of the dye.[10]. Turn on and adjust a spectrophotometer to a wavelength of 595nm, and blank the spectrophotometer using 1.5 mL cuvettes or use a mobile smartphone camera (. [7], The different colours are a result of the different charged states of the dye molecule. and diagrams provided correct acknowledgement is given. Adjust the spectrophotometer to a wavelength of 595nm, using the tube which contains no protein (blank). It is bounded by the provinces of Warmisko-Mazurskie to the north, Podlaskie to the northeast, Lubelskie to the southeast, witokrzyskie to the south, dzkie to the southwest, and Kujawsko-Pomorskie to the northwest. After electrophoresis, incubate 1 or 2 gels in a staining container containing 100 ml Coomassie Blue R-250 staining solution. [20][21] The mobility of the complex in the polyacrylamide gel will depend on both the size of the protein complex (i.e., the molecular weight) and the amount of dye bound to the protein. Before Coomassie blue. You may use any Coomassie staining protocol of choice. Mechanism of Coomassie brilliant blue G-250 binding to proteins: a After incubation, discard the stain. National Library of Medicine Please enable JavaScript Coomassie brilliant blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Fast and sensitive colloidal coomassie G-250 staining for - PubMed [6], In order to measure the absorbance of a colorless compound a Bradford assay must be performed. The suffix "R" in the name of Coomassie brilliant blue R-250 is an abbreviation for "red" as the blue colour of the dye has a slight reddish tint. Anal Biochem. Trap column-based intact mass spectrometry for rapid and accurate evaluation of protein molecular weight. Same test tubes cannot be used since the stain would affect the absorbance reading. This provides a simpler method for fingerprint analysis by reducing the number of amino acids needing to be analyzed from 23 to 6 and requires little to no assay preparation, in contrast to the ninhydrin chemical assay, which requires assay preparation such as heating and enzyme cascade.[32]. Catalog Number 115444. Wait 2 minutes and read the absorbance of each standard and sample at 595nm. Incubate the gel in this staining solution as follows at room temperature with gentle shaking: Tris-Glycine, Tricine, and Zymogram Gels for a minimum of 3 hours and a maximum of 12 hours . Stain cannot be re-used. Coomassie Brilliant Blue R-250 (161-0436) Coomassie Brilliant Blue R-250 staining solution is the fastest and easiest way to Coomassie-stain Criterion precast gels or other polyacrylamide protein gels. Wait 5 minutes and read each of the standards and each of the samples at 595nm wavelength. Use this Coomassie brilliant blue R-250 solution to stain proteins in SDS-PAGE gels. Please call customer service for assistance: 1-800-766-7000. Changes to the original method, such as increasing the pH by adding NaOH or adding more dye have been made to correct this variation. If you need to leave the gel overnight in the stain, add 2 ml of 20% NaCl (w/v) in water for every 20 ml of stain. This site needs JavaScript to work properly. Coomassie brilliant blue G-250 (100 mg) is dissolved in 50 cm 3 95% ethanol. Unauthorized use of these marks is strictly prohibited. Prepare standard concentrations of protein of 1, 5, 7.5 and 10g/mL. Decant staining solution and add a minimum of 200 ml of deionized water per gel to the staining container. Coomassie Brilliant blue R 250 (C.I. R represents the sum of the square values of the fit subtracted from each data point. [7], The dye interacts electrostatically but noncovalently with the amino and carboxyl groups of proteins. HHS Vulnerability Disclosure, Help Thermo Fisher Scientific. Thermo Scientific Pierce Coomassie Brilliant Blue Dyes - Fisher Sci [13] It is an extremely sensitive technique. Add 5.0 mL of Coomassie Blue to each tube and mix by vortex, or inversion. Coomassie brilliant blue G-250, the binding dye for the Bradford Method Color reaction of protein and Bradford reagent The Bradford assay, a colorimetric protein assay, is based on an absorbance shift of the dye Coomassie brilliant blue G-250. The CBB stain forms a strong, noncovalent complex with the carboxyl group of the protein by van der Waals force and the amino group through electrostatic interactions. [7] This is the basis of the Bradford assay, which quantifies protein by Coomassie brilliant blue dye binding. Careers. A high concentration of buffer will cause an overestimated protein concentration due to depletion of free protons from the solution by conjugate base from the buffer. [6], Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemical compounds such as sodium, potassium or even carbohydrates like sucrose, that may be present in protein samples. Our studies show Coomassie Brilliant Blue G-250 as a promising chemical chaperone that stabilises the -helical native human insulin conformers, disrupting its aggregation. Brilliant Blue G Write a review pure Synonym (s): Acid blue 90, Coomassie Brilliant Blue G Empirical Formula (Hill Notation): C47H48N3NaO7S2 CAS Number: 6104-58-1 Molecular Weight: 854.02 Colour Index Number: 42655 Beilstein: 5230822 EC Number: 228-058-4 MDL number: MFCD00078482 PubChem Substance ID: 24891535 NACRES: NA.47 Papers published in biochemistry journals frequently refer to these dyes simply as "Coomassie" without specifying which dye was used. This procedure will not affect sensitivity. The reagent is prepared as follows. Coomassie Brilliant Blue R-250, Fisher BioReagents Loosely cover the staining container and heat in a microwave oven at full power for 1 minute. sharing sensitive information, make sure youre on a federal At pH7 the dye has an extinction coefficient of 43,000M1cm1. In making these dilutions, error in one dilution is compounded in further dilutions resulting in a linear relationship that may not always be accurate. Commun., 2023, Accepted Manuscript Dissolve the 3g of Coomassie Dye in 450mL methanol. QC Colloidal Coomassie (161-0803) the newest in the family of Bio-Rad Coomassie stains, QC Colloidal Coomassie G-250 allows for flexible staining and destaining times and does not require use of methanol for fixing. Please enable it to take advantage of the complete set of features! [14][15][16][17], The Bradford assay uses the spectral properties of Coomassie brilliant blue G-250 to estimate the amount of protein in a solution. The green colour corresponds to a form of the dye with no net overall charge. Howard Hughes Medical Institute/United States. Step 1: Stain gels for 12 hours with gentle agitation. Unauthorized use of these marks is strictly prohibited. Created in 1999 as one of 16 new provinces, it comprises the former . [28][29][30], Tissueblue was approved for medical use in Canada in January 2021. Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models, Rapid coomassie blue staining of protein gels, Inverted Light Microscope: A Comprehensive Guide for Students of Microbiology and Laboratory Technicians, DIY Home CRISPR: A comprehensive how to guide, Solubility in water; Slightly soluble in cold, and soluble in hot (bright blue). You may use any Coomassie staining protocol of choice. Two main types of coomassie staining exist: The original or "classical" coomassie; The newer colloidal coomassie. If nucleic acids are present in the sample, they would also absorb light at 280nm, skewing the results further. Description Use this Coomassie brilliant blue R-250 solution to stain proteins in SDS-PAGE gels. The final proton is lost under alkaline conditions and the dye becomes pink (pKa 12.4). There is a small amount of dye in the water that is in equilibrium with the dye bound to the protein, so proteins will remain blue. Polski: Wojewdztwo mazowieckie jest wojewdztwem w centralnej Polsce nad rzek Wis ze stolic w Warszaw. Prepare staining solution for a single gel as described in the table below. The ionic interaction further strengthens the bond. Bio-Rad offers Coomassie stains in four formats. Brilliant Blue G pure 6104-58-1 - MilliporeSigma SDS. This requires spectrophotometers capable of measuring in the UV range, which many cannot. The "250" originally denoted the purity of the dye. Furthermore, it is completely compatible with mass spectrometric protein identification. This complex formation enables the detection of the proteins separated by the gel. In summary, in order to find a standard curve, one must use varying concentrations of BSA (Bovine Serum Albumin)[2] in order to create a standard curve with concentration plotted on the x-axis and absorbance plotted on the y-axis. The anionic bound form of the dye which is held together by hydrophobic and ionic interactions, has an absorption spectrum maximum historically held to be at 595 nm. If you need to store the gel in water for a few days, add 20 ml of 20% NaCl. By using the Bradford protein assay, one can avoid all of these complications by simply mixing the protein samples with the Coomassie brilliant blue G-250 dye (Bradford reagent) and measuring their absorbances at 595nm, which is in the visible range[8] and may be accuretaly measured by the use of a mobile smartphone camera. Different kinds of Coomassie dyes are available. The stain transfers an overall negative charge to the proteins allowing their separation from the polyacrylamide gel. Click here As an Amazon Associate Conductscience Inc earns revenue from qualifying purchases The modern pipette has had a colorful history, Introduction CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats which are an important part of the bacterial defense system and form the bases, Scientific investigations, preclinical research, and pharmacological studies use a number of laboratory animals as subjects. Safety Data Sheet for Coomassie Brilliant blue G 250 (C.I. Bio-Rad offers Coomassie stains in three major formats. Nagata K, Ashikaga R, Mori W, Zako T, Shimazaki Y. Anal Sci. A microscope is an essential tool that is used in most laboratories. Coomassie Brilliant Blue R-250 (161-0436) Coomassie Brilliant Blue R-250 staining solution is the fastest and easiest way to Coomassie-stain Criterion precast gels or other polyacrylamide protein gels. The https:// ensures that you are connecting to the Protein ranging from 2.5 ng to 100 ng could be identified using the Coomassie brilliant blue stain. Coomassie Brilliant Blue R-250 0.05 g 0.05%: Methanol 50 mL: 50% (v/v) Glacial acetic acid 10 mL: 10% (v/v) H 2 O to 100 mL: Dissolve the Coomassie Brilliant Blue R-250 dye, and then filter through a Whatman No. The Bradford assay is linear over a short range, typically from 0g/mL to 2000g/mL, often making dilutions of a sample necessary before analysis. You do not have JavaScript enabled. 2022 Aug;2(8):e527. Search 1 L, Coomassie Brilliant Blue R-250 staining solution, The minimum orderable quantity of this product is 1, Protein Expression / Characterization / Quantitation, Blood Typing, Screening & Antibody Identification, Genetic Engineering, Microbiology & Model Organisms, contact your local sales office or representative, Coomassie Brilliant Blue R-250 Staining Solution, Mixture of water, methanol, and glacial acetic acid, Coomassie Brilliant Blue R-250 staining solutions kit (, Coomassie Brilliant Blue R-250 powder, 10 g (, Coomassie Brilliant Blue G-250 powder, 10 g (. Gently shake gel in water for at least 7 hours. The R-250 (red-tinted) form lacks two methyl groups that are present in the G-250 (green-tinted) form, which is also called colloidal coomassie dye. Coomassie brilliant blue G-250 (CBBG-250), known as a chemical dye, has a molecular formula of C 47 H 48 N 3 O 7 S 2 Na whose structure is shown in Fig. Coomassie R-250 and G-250 dyes are two chemical forms of a disulfonated triphenylmethane compound that is commonly used as the basis of stains for detection of proteins in gel electrophoresis and Bradford-type assay reagents for protein quantitation. Two years later in 1965 Meyer and Lambert used Coomassie brilliant blue R-250 to stain protein samples after electrophoretic separation in a polyacrylamide gel. Coomassie Brilliant Blue removal/disposal from gel destain and used gel stain in an environment-friendly manner. Agudelo., & Halmek., J. [Na+], Except where otherwise noted, data are given for materials in their, 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, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding", "Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury", "Blue Food Dye Treats Spine Injury in Rats", "TissueBlue- brilliant blue g injection, solution", "Summary Basis of Decision (SBD) for Tissueblue", 4'-O--D-Glucosyl-9-O-(6''-deoxysaccharosyl)olivil, https://en.wikipedia.org/w/index.php?title=Coomassie_brilliant_blue&oldid=1141968865, Pages using collapsible list with both background and text-align in titlestyle, Articles containing unverified chemical infoboxes, Short description is different from Wikidata, Wikipedia articles needing clarification from January 2022, Creative Commons Attribution-ShareAlike License 3.0, Insoluble in cold, slightly soluble in hot (bright red blue), Slightly soluble in cold, soluble in hot (bright blue), This page was last edited on 27 February 2023, at 20:36. official website and that any information you provide is encrypted After mixing well, the mixture almost immediately changes to a blue color. [Protein determination by binding with the dye Coomassie brilliant blue G-250]. The dye is noted for its high level of sensitivity: 5 g of protein[clarification needed] can be detected. To perform the assay, x cm 3 of the sample containing 5-100 g of protein is placed in a clean, dry test tube. Oh., J. H. Change destaining solution (1610438) multiple times (e.g., 4 washes x 30 min) until the background is less dark. Environmentally safe removal/disposal of Coomassie Brilliant Blue from gel destain and used gel stain. Mechanism of Coomassie Brilliant Blue G-250 binding to cetyltrimethylammonium bromide: an interference with the Bradford assay. On binding to a protein, the negatively charged Coomassie brilliant blue G-250 dye molecule will give an overall negative charge to the protein. Some colorless compounds such as proteins can be quantified at an Optical Density of 280nm due to the presence of aromatic rings such as Tryptophan, Tyrosine and Phenylalanine but if none of these amino acids are present then the absorption cannot be measured at 280nm. As the dye stained the polyacrylamide gel as well as the protein, in order to visualise the protein bands they needed to destain the gel, which they did electrophoretically. Copyright 2006-2023 Thermo Fisher Scientific Inc. All rights reserved, Colonnes et cartouches de chromatographie, Consommables en plastique et fournitures de laboratoire, Afficher toutes les catgories de produits, Spectroscopie, analyse lmentaire et isotopique, Voir toutes les applications et techniques, Services aux organisations de dveloppement et de fabrication sous contrat (CDMO) et pour les essais cliniques, Consultez toutes les rubriques d'aide et d'assistance, 0.1% Coomassie R-250 in 40% ethanol and 10% acetic acid (if using Invitrogen Coomassie R-250 Staining), Destaining Solution consisting of 10% ethanol and 7.5% acetic acid (if using Invitrogen Coomassie R-250 Staining). Animal Models for the Investigation of P2X7 Receptors. This method can also make use of a Vis spectrophotometer[16] or a mobile smartphone camera (RGBradford method).[9]. 1 L, ready-to-use, non-hazardous colloidal Coomassie G-250 stain for protein polyacrylamide gels, The minimum orderable quantity of this product is 1, 1 L, premixed staining solution, for polyacrylamide protein gels, 5 L, premixed staining solution, for polyacrylamide protein gels, Kit for staining protein-containing polyacrylamide gels, includes 1 L Coomassie Brilliant Blue R-250 staining solution and 2 x 1 L destaining solution, 1 L, Coomassie Brilliant Blue R-250 staining solution, 4 x 1 L, Coomassie Brilliant Blue R-250 staining solution, 1 L, Coomassie Brilliant Blue R-250 destaining solution, 4 x 1 L, Coomassie Brilliant Blue R-250 destaining solution, 10 g, Coomassie Brilliant Blue R-250 protein stain powder, 10 g, Coomassie Brilliant Blue G-250 protein stain powder, Instructions for Staining Polyacrylamide Gels, Rev B, QC Colloidal Coomassie Stain Manual, Rev A, Protein Expression / Characterization / Quantitation, Blood Typing, Screening & Antibody Identification, Genetic Engineering, Microbiology & Model Organisms, Ready-to-use Coomassie Brilliant Blue R-250 staining and destaining solutions (161-0435), Staining and Visualization of Proteins After 2-D Electrophoresis, Imaging and Analysis of 2-D Electrophoresis Gels, Coomassie Brilliant Blue R-250 Staining Solutions Kit, Coomassie Brilliant Blue R-250 Staining Solution, Coomassie Brilliant Blue R-250 Destaining Solution, contact your local sales office or representative, Low background, high sensitivity, superior reproducibility, Environmentally friendly formulation no addition of methanol or acetic acid required; eliminates the need for hazardous waste disposal, Flexible staining and destaining times from 1 hour to overnight, No alcohol addition or dilution steps when staining polyacrylamide gels, One-part, ready-to-use colloidal Coomassie stain, Premixed, ready-to-use, nonhazardous solution, No methanol or acetic acid required for destaining, Bio-Safe composition reduces solvent waste disposal costs. Lab Anim Res. When the dye binds to the protein, it causes a shift from 465nm to 595nm, which is why the absorbance readings are taken at 595nm.