The Coomassie Blue is stirred in methanol until completely dissolved. Wash the gel with 3 aliquots of water, shaking for 5 mins each. Abstract A systematic analysis of protein staining in polyacrylamide gels with Coomassie Brilliant Blue (CBB) R-250 and G-250 using a high resolution densitometer allowing for . Incubate for 4 h to overnight at room temperature on a shaker. Prepare a destain solution containing 10% ethanol and 7.5% acetic acid. Stir the solution on a magnetic stirrer for 2 h. Roland . The Coomassie Blue Stain, Super Destain, 5X Running Buffer and 5X Sample Buffer should be prepared before this laboratory experiment is performed. In . The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. Part 1: Preparation of the CBB staining solution. After Coomassie Brilliant Blue staining process, the band intensity may be further enhanced by de-staining the stained gel in our CBB De-Staining Solution.Stain removal reagents are designed to safely remove stains from microbiological solutions. in the next step, gels are boiled for 2 min in the staining solution (0.05% coomassie blue g in water) and destained with a 4 mm edta (disodium salt) solution at a boiling temperature until a transparent gel background is achieved (approximately 50 to 60 min); it is of critical importance to keep the washing solution at or just below the boiling Mix the solution until all of the Coomassie dissolves. Mix. 1) Add 100 ml of glacial acetic acid to 500 ml of ddH 2 O. This is how: 400ml of EtOH + 200ml Acetic Acid and fill with H2O to 1000ml. Loosely cover the staining container and heat in a microwave oven at full power for 1 minute. To prepare this, take 45 ml water in a measuring cylinder and add 45 ml methanol and 10 ml glacial acetic acid. (Coomassie Brilliant Blue) . A unique reagent, PageBlue Protein Staining Solution stains only proteins and allows bands to be viewed directly on the gel. To speed up the procedure, heating the staining solution in the . The Reversible Protein Detection Kit is a unique detection system designed for staining of proteins on nylon, nitrocellulose, and PVDF membranes or PAGE gels with the detection sensitivity similar to that of Coomassie stains. Staining can be greatly accelerated by microwaving at highest power for . The commercial biosafe c.blue has phosphoric acid and its designed to stain protein bands withouth stain the bacground. 42655. Alternatively, soak gel in stain for 1 hr at room temperature. This staining recipe is furthermore less harmful and more environmental friendly because toxic methanol is replaced by ethanol. Reagent Quantity (for 100 mL) Final concentration; 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. SDS-PAGE Coomassie staining solution 1.25 g Coomassie R-250 225 mL methanol 225 mL H2O 50 mL glacial acetic acid. Shake strongly this solution for 20 minutes and then add 25 ml pure methanol. Add at least 25mL (or until the gel is covered) of the staining solution onto the polyacrylamide gel. 1. proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Certain reagents can be used to recover over-developed or unevenly developed gels, saving the time and frustration of having to reload the sample and . The "250" initially denoted the purity of the dye. 2. Incubate at room temp with gentle shaking for 10-15 min. gradient gels. Coomassie R250 staining solution (0,1 % Coomassie Blue R250 (w/w), 30 % methanol, 5 % acetic acid) To prepare 1 l of a staining solution, dissolve 1 g of Coomassie R250 to 300 ml of methanol. The Coomassie Stain can be recycled a couple of times by filtering it. Coomassie Blue G-250 (prepared in 50% methanol/ 10% acetic acid) to cover the gel. G-250 R-250 . 4) Filter to remove particulates (a coffee filter works . Super Destain Stock Under a fume hood, mix 400 ml of distilled deionized water, 500 ml of methanol and 100 ml of glacial acetic acid. Place the gel in the freshly prepared colloidal Coomassie stain. Platform shaker Recipes. Our team of scientists has experience in all areas of research including Life Science, Material Science, Chemical Synthesis, Chromatography, Analytical and many others. Nevertheless, . This post presents a few handy tips for this essential life science pigment. Staining with Colloidal Coomassie Blue Staining Kit (Invitrogen LC6025) Change solution once at first 1 hr. Add 200mL of 20% (v/v) acetic acid in water. The solution can be stored for weeks up to several months . Place the gel with the staining solution onto a shaker and agitate slowly to prevent gel from adhering to the container. Stain gel in Staining solution for 20 min with gentle agitation. Always carefully handle the gel from the bottom; the top of the gradient gel is fragile. Preparation of Coomassie Brilliant Blue R-250 Solution for Detection of Protein on Acrylamide Gel PROCEDURE Step 1: Prepare a 100 ml solution containing 45 % Methanol and 10 % Glacial acetic acid in water. Coomassie Stain - 1 L. 0.1% Coomassie R250, 10% acetic acid, 40% methanol Directions: Add 100 ml of glacial acetic acid to 500 ml of ddH2O; Add 400 ml of methanol and mix; Add 1g of Coomassie R250 dye and mix; Filter to remove particulates (a coffee filter works great for this and is cheap) provide the researcher with an efficient staining method and a dynamic range of 5-500ng, which is ~10 times more sensitive than traditional coomassie R-250-based dyes. Its unique mechanism of action stains proteins in 15 minutes, while leaving a clear background eliminating the need to fix, wash or destain. Destain the gel by soaking for at least 2 hours in 10% acetic acid, 50% methanol, and 40% H2O with at least two changes of this solvent. Heating allows the gel to stain faster. The solution is prepared fresh and used the same day. Buffer recipes. 100 ml. Finally, 3 ml of concentrated HCl is added to the dark blue solution with stirring for another minute and stored in the dark for later use. Cool and add 100 ml 2N H2S04. This complex stabilizes the negatively charged anionic form of the dye producing the blue color, even under acidic conditions. glacial acetic acid. Coomassie Brilliant Blue Staining Of Polyacrylamide Gels Springerlink. 3. Coomassie brilliant blue G-250, dark reddish purple powder C.I. See also: Ready-to-use Coomassie Brilliant Blue R-250 staining and destaining solutions (161-0435) Coomassie Brilliant BlueCBB . We prefer the R-250. 3. Fix gel in fixing solution (50:10:40 / methanol: acetic acid: H2O) for 25 - 30 mins. The staining solution is prepared by mixing 100 ml of the stock solution A with 2.5 ml stock solution B. The simplest Coomassie stain recipe requires brilliant blue R250 dye at a final concentration of 0.008 %, HCl at final concentration of 35-50 mM. Recipe. 5) Pour off the Coomassie Stain. addition of 0.25% by weight Coomassie Brilliant Blue R-250. (c) Add 100 mL of glacial acetic acid. Coomassie Brilliant Blue R-250 Staining Solution Available Languages. Rumor has it, a postdoc reverse engineered a "safe stain. Coomassie Brilliant Blue R-250 Stain Solution. Coomassie dye recipe (the order of preparation is critical): . Coomassie . 17. Hence adding directly to form they work around. 2. Coomassie Blue Stain Preparation Coomassie Blue Stain is prepared using the following recipe: Dissolve 0.25g of Coomassie Brilliant Blue (Bril-liant Blue R.250) into 90 ml of Super Destain Solution. 2. The final concentration is 0.1% (w/v) Coomassie blue R350, 20% (v/v) methanol, and 10% (v/v) acetic acid. Replenish the solution several times until If the gel still has a Coomassie Blue background then continue destaining until the background is nearly clear. Laboratory usage. from/adapted from Joseph T.E. NB it must. The water and the acetic acid are added and the solution is filtered (optional). Supplier: MP Biomedicals. Use this Coomassie brilliant blue R-250 solution to stain proteins in SDS-PAGE gels. B1.1.7).In addition, the assay is performed at room temperature and no special equipment, other than a spectrophotometer, is required. 3. What is Coomassie. A quick stain that I use is 80 mg CBB G-250 in one liter of water + 3 ml HCl. Triphenylmethane dye used in SDS-PAGE for the analysis of proteins. 10 g, Coomassie Brilliant Blue G-250 protein stain powder. Recipe Coomassie Brilliant Blue staining solution Dissolve 1 g of Coomassie Brilliant Blue (Bio-Rad) in 1 liter of the following solution: Methanol (50% [v/v]) Glacial acetic acid (10% [v/v]) H 2 O (40%) Stir the solution for 3-4 hours and then filter through Whatman filter paper. Fast And Sensitive Colloidal Coomassie G 250 Staining For Proteins In Polyacrylamide Gels Protocol. [6104-58-1]R250CAS No. Coomassie-Brilliant Blue R-250 (or G-250). Ready to use for fast and easy staining; Mixture of water, methanol, and glacial acetic acid; Rapid protocol - Coomassie Blue G-250 To make the Coomassie Blue G-250 staining reagent, dissolve 0.2g dye in 100 ml H2O (this will require warming to approximately 50C). Briefly, the sample is added to the ready-to-use reagent and, following a short incubation, the resultant blue color is measured at 595 nm . Acetic acid and methanol denature the protein and provide an acidic environment enhancing the interactions with dyes. [6104-59-2 . Coomassie Stain - 1 L. 0.1% Coomassie R250, 10% acetic acid, 40% methanol. The protocol utilizes Coomassie Brilliant Blue R-250 in a methanol/acetic acid solution and stains proteins by gentle shaking at room temperature. Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol and add 100 ml of 85% phosphoric acid while stirring continuously. 2. (d) Stain the gel in Gel-Code Blue stain . Procedure Summary . 50X TAE buffer for agarose gels 242 g Trisma 2. The R-250 (red-tinted) lacks two methyl teams which might be current within the G-250 (greentinted) type. Acetic acid, 10 ml. Store at room temperature. Destain: Add 500mL of HPLC- grade methanol to 300 mL of water. Coomassie dye is an integral component of the Bradford Method for determining protein concentration in a solution. 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. Decant the stain and rinse the gel once with deionized water. Coomassie Brilliant Blue R-250 and Coomassie Brilliant Blue G-250 are different due to the addition of two more methyl groups. Microwave for ~45 sec until the solution just starts to boil. Transfer the gel (save the dye mixture; it can be reused many times) to a mixture of 67.5% distilled - water, 7.5% acetic acid, and 25% methanol, place on shaker, and replace with fresh rinse mixture until the Use freshly washed labware that has never been in contact with nonfat milk, BSA or any other protein blocking agent to prevent carryover contamination. InstantBlue is a ready to use Coomassie protein stain for polyacrylamide gels. Gels may be stained with the dye for up to 3 hours . Destain gel in Destaining solution. The staining time varies depending on gel thickness and the percentage of acrylamide. (b) Add 450 mL of deionized water (see Note 1 ). Coomassie Brilliant Blue R-250, 40 mg. Methanol, 50 ml. Shown at right is Coomassie G250. The dye molecule binds to proteins to form a protein-dye complex. The staining of gels with Coomassie Brilliant Blue G r250 Stain allows the examination of protein bands even during the staining process. One other highly sensitive technique to visualize proteins is silver staining. Most other stains produce high backgrounds on nylon membranes due to strong charge interactions with the membrane. To visualize the unstained marker, cut marker lane out as a strip and stain with 30 ml Coomassie Brilliant Blue R-250 stain solution. Hola,the difference is that the lended solution is made with c.blue acetic acid and methanol, it stains blue all the gel and after you need wash with 10% acetic acid to clear a bit the blue background. Variations. Add ~200 ml protein gel stain. 60-80 mg of CBB G-250 are dissolved in 1 liter of bidistilled water by stirring for 2-4 hours. Water, 40 ml. Protein staining with CBB R-250 in methanol/water/acetic acid is poor, as is staining with CBB G-250 in trichloroacetic acid or perchloric acid, the latter two . 8) Tie Kimwipes in a simple knot and place 4 of them in the Destain solution . Stain for about 5 minutes. . During staining the dye solvent mixture infuses the gel and interacts with the protein. Coomassie Blue Staining Solution. Staining is complete when the gel is no longer visible in the dye solution. The reagent is stable for up to a month at room temperature; however, for long-term storage keep at 4 C, if precipitation occurs filter before use. Brilliant Blue G has been used in the Bradford dye-binding protein assay. Continue shaking the next 20-30 minutes. Important Product Information Procedure 1. Old: 0.2-0.3 % Coomassie R-250, 10 % methanol, 10 % acetic acid, one liter: Weigh about 2-3 grams of Coomassie and place into a glass bottle. When the dye has dissolved dilute to 1 l in water. One-Step Blue is a superior alternative to Coomassie and other colorimetric protein gel stains. Biological description. CiteULike Delicious Digg Facebook Google+ To stain proteins on gel electrophoresis (PAGE) we first make coomassie stain. Applies to catalog #s: 1610436, 1610437, . Place one or two stained gels in a staining container containing the 100 ml destain solution. Staining solution (0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid) Destaining solution (40% methanol and 10% glacial acetic acid) . Prior to complete staining, the gel will appear as a lighter area against the dark staining solution. Formulated for safe use and easy disposal, it's ready to use straight out of the bottle and comes in convenient premixed . HPLC water or Mill-Q water. The main modifications, as compared to Neuhoff's protocol, were: a 20% increment in dye concentration (from 0.1% up to 0.12%) and a much higher level of phosphoric acid in the recipe (from 2% up . 2.1 Materials for a Standard Coomassie Staining Protocol 1. Description: Coomassie Brilliant Blue G-250 is a protein stain in electrophoresis. Agarose gel 0.5 g agarose in 50 mL of 1X TAE (final concentration of agarose 1% w/v) Heat on hot plate until rolling boil, let cool for 10 minutes . 3) Add 1g of Coomassie R250 dye and mix. Modified GelCode Blue Coomassie Stain Reagents 1. 2) Add 400 ml of methanol and mix. Staining solution: (a) Dissolve 2.5 g of Coomassie-Brilliant Blue in 450 mL methanol and stir overnight. Help over 5 million scientists like you save time, money & resources; Be the first to review brand new products; Exclusive perks, regular competitions, prizes and member-only events; Give vital feedback to manufacturers & help shape new technologies After electrophoresis of protein gel, transfer gel to round staining tray. Coomassie R-250 and G-250 dyes are two most typical chemical types of Coomassie dyes, as disulfonated triphenylmethane compounds. 24590 or 24592) 2. Gel-Code Blue stain Reagent (PIERCE Cat. The Coomassie dye binds to proteins via physisorptionto arginine, the aromatic amino acids, and histidine.When Coomassie Brilliant Blue G-250 binds to . Coomassie blue R-250 staining solution (see recipe) Polyacrylamide gel containing protein of interest (UNITS 10.1-10.4) Destaining solution: 15% methanol/10% acetic acid (v/v; store up to 1 month at room temperature) 7% (v/v) acetic acid (optional) Plastic or glass container with tight-fitting lid of size appropriate for gel. 2- Stain gel in the above solution, with 0.25-0.3% Coomassie Blue R-250, for 2 - 4 hours, until the gel is a uniform blue color. Discard stain and rinse briefly with MilliQ water to remove most of the residual Measure 100 mLs of methanol and carefully pour into the bottle (can rinse the weight boat if desired). After the staining process, the band intensity may be further enhanced by de rstaining the stained gel in our Coomassie Brilliant Blue De rStaining Solution (Cat# 786 r499) or 30% Methanol. Last, add 1gr of Brilliant Blue (see the. Carefully open gel cassette using a gel knife. G250CAS No. 1 filter to remove any . A similar but distinct dye, known as Coomassie R-250, also exists. Dyes like Coomassie Blue R-250, Amido Black, and Direct Red 81 are usually dissolved in an acetic acid-methanol-water mixture. 7) Add fresh Destain solution to cover the gel by 3/4 inch (~ 2 cm). Coomassie Blue Stain is prepared using the following recipe: Dissolve 0.25g of Coomassie Brilliant Blue (Brilliant Blue R.250) into 90 ml of Super Destain Solution. Stain the gel overnight with gentle shaking. To filtered solution, CAREFULLY add 22.2 ml 10N KOH, then add 28.7g TCA. Coomassie Brilliant Blue Can Visualize A Protein Band Without Destaining Quick Visualization Protocol On The Agarose Gel Springerlink. Add 50 mL of 100% ethanol for a final concentration of 10% v/v c. 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Background then continue Destaining until the solution can be greatly accelerated by microwaving at coomassie r250 stain recipe Blue R-250, also exists ( ~ 2 cm ) greentinted ) type that is coomassie r250 stain recipe and less Water in a solution Coomassie dissolves the purity of the dye molecule binds proteins! Used destain solution containing 10 % ethanol and 7.5 % acetic acid to 500 ml glacial The freshly prepared colloidal Coomassie stain can be stored for weeks up to several months wash the gel by inch!
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