coomassie blue staining sensitivity

The sensitivity is 2-300 times higher than conventional Coomassie Blue staining and ca. Although traditionally used for densitometry, CBB has fluorescent properties. This can be short-ened to a minimum of 1 h staining time, After staining with the dye, you will observe blue bands with a blue background. 1. Faint bands on a high background. It is the most commonly used staining technique for the quantification of proteins after gel electrophoresis. The goal here isnt to purify the proteins, instead the goal is just to see whats there. Discard stain and rinse briefly with MilliQ water to remove most of the residual Detection sensitivity is approximately 20% that of Coomassie Blue; Providing the highest sensitivity, the ProteoSilver Silver Stain Kit has a sensitivity of 0.1 ng of protein per band with an incubation time of 3-12 minutes. Coomassie R-250, and its dimethylated derivative G-250, have been used as protein gel stains for more than 45 years. Gels were sensitized with SYPRO Ruby and then stained with the Coomassie Brilliant Blue G-250 protocol (Blue Silver). Dye that is not bound to protein diffuses out of the gel during destain steps. Another advantage of Coomassie Blue stain is that it doesnt chemically modify proteins in SDS-PAGE, so it is compatible with downstream applications like mass spectrometry and peptide sequencing. Some premade and traditional homemade Coomassie R-250 protein stains can take three hours or more to fully stain gels, and then require destaining typically overnight. The method is based on staining with Colloidal blue dye in methanolic solution with phosphoric acid and high ammonium sulfate concentration. Moreover, when a combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. Molecular formula. The gel should be exposed to 10% acetic acid, 50% methanol for a total (stain plus destain) period of at least 3 hours (with shaking and at least Smeared or blurred bands. Coomassie Blue Stain Solution 1x Strength Solution: The Ultra-Sensitive Coomassie Blue Stain Solution, based on Coomassie dye (G-250), is an enhanced protein stain that offers unsurpassed sensitivity and rapid band visualization. Supplied in a ready-to-use format of 1XStrength Solution, the staining solution is The proteins are detected as blue spots or bands on a clear background. A novel method for revealing proteins after electrophoresis on cellulose acetate is described, based on adsorption of mixed gold-Tween 20 micelles onto TCA-fixed protein bands. Perhaps the best known in-gel detection method is the ubiquitous Coomassie Blue (CB) stain; CB has served as a gel stain and protein quantification reagent for over 40 years. during the staining process. This combined Electrophoresis 6(9):427448 Candiano G, Bruschi M, Musante L et al (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. One common way to use it is to dissolve the dye in a mixture of methanol, acetic acid, and water. Sign In. There are several reasons: Low price. Coomassie G-250 staining solution with an optimized staining protocol to achieve the highest sensitivity of any commercially available Coomassie stain. Save time and money by having us create a batch of 10X Tris-Glycine-SDS Buffer for you! - Coomassie (non-colloidal) staining: Stain in 0.1% Coomassie Blue R-250 in 50% methanol for 5 minutes and destain with several changes of 50% methanol and 10% acetic acid. Though less sensitive, Coomassie G-250 can be used in place of the R-250 form to create a rapid and convenient staining procedure. 1. The protocol involves soaking the gel in a dye solution. After incubation, discard the stain. Sambrook et al. Sambrook et al. 1 Improvements over the years have increased sensitivity, and Coomassie staining is compatible with downstream analysis by mass spectrometry (MS). Coomassie Brilliant Blue is commonly used for the detection of proteins in sodium dodecyl sulfatepolyacrylamide gel electrophoresis, owing to its reliability and simplicity. Coomassie Blue G-250 (prepared in 50% methanol/ 10% acetic acid) to cover the gel. Realizing the limitations of both the classical and colloidal Coomassie stains, enhanced Coomassie dyes such as RAPIDStain TM and LabSafe GEL Blue TM have been formulated to address the perceived gaps. The Coomassie dyes (R-250and G-250) bind to proteins through ionic interactions between dye sulfonic acid groups and positive protein amine groups as well as through Van der Waals attractions. With a marker, place ethanol and lint-free cloths before use u0007 dd 510 l of Reagent A' to each tube A Add 127 l of Reagent A' to each tube 3 a mark on the glass plate 1 cm below the teeth of the comb. For greater sensitivity and reduced background, gels can be stained for 1 hour and de-stained overnight in water. Coomassie blue dyes bind proteins quantitatively within a certain protein range allowing for densitometry analysis. PageBlue protein stain can deliver a dynamic range of ~5ng to ~500ng. Silver staining has greater sensitivity, but involves many more steps and solutions (see Silver Staining of SDS-polyacrylamide Gel ). (1989). The related fixative comprises an acid and an alcohol, wherein the alcohol is methanol and/or ethanol, and the acid is an acid mixture of acetic acid phosphoric acid in a volume ratio of 1:1. The binding of the dye to a protein causes a shift in the absorbance One of the most commonly used, Coomassie Blue G-250 can be used as follows; after electrotransfer, wash the polyacrylamide gel three times with ddH2O. The related staining agent comprises 0.05-0.12% by mass/volume 1. To obtain the clearest background for photography, perform a second 1-hour wash with 100 mL of water. Coomassie G-250 manifests a leuco form below pH 2. Bio-Safe Coomassie (161-0786) Bio-Safe Coomassie Brilliant Blue G-250 stain is fast, simple, sensitive, and convenient. Coomassie Brilliant Blue has the potential to detect 30-100 ng proteins. Product Description: Coomassie Blue Stain Solution (Protein Stain-EZ A Ultra Sensitive) 1x Strength Solution: The Ultra-Sensitive Coomassie Blue Stain Solution, based on Coomassie dye (G-250), is an enhanced protein stain that offers unsurpassed sensitivity and rapid band visualization. Disadvantages: complicated procedures and high cost. Smeared or blurred bands. 1- 100ml fixing solution : 50 % Methanol, 10% Acidic Acid, 50 l Formalaldehyde It offers a sensitivity that is better than conventional Coomassie R-250 formulations and equivalent to Coomassie Blue G-250, but with a simpler and quicker staining protocol. 1- 100ml fixing solution : 50 % Methanol, 10% Acidic Acid, 50 l Formalaldehyde In spite of the high sensitivity of silver staining and the wide dynamic range of various fluorescent detection methods, Coomassie Brilliant Blue staining is still the most widely used protein detection technique for proteins separated by polyacrylamide gel electrophoresis. This can be short-ened to a minimum of 1 h staining time, Neuhoff V, Stamm R, Eibl H (1985) Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: a systematic analysis. The product can be stored for up to 12 months. However, its lengthy processing time is a major drawback of the sta- n dard Coomassie staining technique. Don't have a profile?Register 100 ng for Coomassie Brilliant Blue R-250, about 30 ng for Colloidal Coomassie Brilliant Blue G Storage instructions. Coomassie Brilliant Blue R r250 Stain has a sensitivity of staining 8r10ng protein/band, i.e. Enhanced Coomassie Stain. Bands will begin to develop within minutes. The formation of the complex stabilises the negatively charged anionic form of the dye, producing the blue colour, even under acid conditions when most of the molecules in solution are in the cationic form. Note: Gel can be stained for up to 3 hours, but after 3 hours, sensitivity will decrease. This is the basis of the Bradford assay, which quantifies protein by Coomassie brilliant blue dye binding. Filter to remove any precipitates 5. Stain the gel with 0.1% (or less) Coomassie Blue R250 in 10% acetic acid, 50% methanol, and 40% H2O for the minimum time (typically less than one hour) necessary to visualize the bands of interest. A common gel stain, Coomassie blue protein stain can be used for sensitive, quantitative protein detection in gels, with a linear range from ~10 ng to 20 g. For these reasons, most researchers favor the Coomassie staining technique instead. Supplied in a ready-to-use format Both products offer high sensitivity (4 to 8 ng) and produce sharp results within a shorter period of time. The dye is non-toxic, and one of its derivatives used in Europe as a food dye. One of the most popular methods to stain a protein gel is with Coomassie blue. To render these bands visible, the gel matrix needs to be treated with a protein-specific, dye-binding or color-producing chemical such as silver stain or coomassie brilliant blue stain. This will u0007 u0007The height of the stacking gel 6 6 n and vortex. Fluorescent in-gel protein stains are generally preferred for higher sensitivity, reduced background, and wider dynamic range. If you require the sensitivity of silver stain, you probably do not have enough protein for MS analysis. Store under desiccating conditions. Coomassie Brilliant Blue R-250 b- e cause of its relatively high sensitivity (3,6). colloidal coomassie blue (CCB) or SYPRO Ruby (SR), in addition to many others). Change the destaining solution for 3-5 times untill you can see clear bands with almost no blue background. 1 It has been shown to be more sensitive than white-light imaging of Coomassie staining. Acidic and ammoniacal silver It is the method of choice if SDS is used in the electrophoresis of proteins, and is sensitive for a range of 0.5 to 20 micrograms of protein. After the staining process, the band intensity may be further enhanced by de rstaining the stained gel in our Coomassie Brilliant Blue Destaining Solution (Cat# 786 r499) or 30% Methanol. Description. C 45 H 44 N 3 NaO 7 S 2. Notably, several of the Coomassie stain formulations provided infrared fluo-rescence detection sensitivity to <1 ng of protein in-gel, slightly exceeding the performance of Sypro Ruby. The main characteristics for an efficient stain are high sensitivity, low background, large linear range, and ease of use. Coomassie Brilliant Blue R 250 is the most commonly used staining procedure for the detection of proteins. Here, we report dramatically decreased protein staining and destaining time, as well as significantly increased detection sensitivity with the application of enhanced heat. Fix and develop. This capability of G-250 is due to its particular properties. 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 The ProtoGel Sample Prep Kit gives you the sharpest bands possible. In spite of the high sensitivity of silver staining and the wide dynamic range of various fluorescent detection methods, Coomassie Brilliant Blue staining is still the most widely used protein detection technique for proteins separated by polyacrylamide gel electrophoresis. 24590 or 24592) 2. Previews 2(1), 10-11. Rapid, sensitive, and safe Coomassie G-250 stain for protein visualization Catalog Numbers LC6060 and LC6065 To achieve maximum sensitivity, see Stain gels: Maximum sensitivity protocol on page 2. This post presents a few handy tips for this essential life science pigment. ProtoGel Quick-Cast: ready to run in 20 minutes! Why is Coomassie Blue a good stain for proteins? This step enhances stain sensitivity, as weak protein bands continue to develop. sensitive than silver staining, Coomassie Blue staining is a relatively simple and more quantitative method. Supplied in a ready-to-use format of 1XStrength Solution, the staining If the colour of Coomassie, as it says on the jar, is brilliant blue, although it has a different chemical structure, Ponceau can be described as brilliant red.. Protein Stain-EZ A Ultra Sensitive Coomassie Blue Stain Solution 1x Strength Solution: The Ultra-Sensitive Coomassie Blue Stain Solution, based on Coomassie dye (G-250), is an enhanced protein stain that offers unsurpassed sensitivity and rapid band visualization. A modified Neuhoff's colloidal Coomassie Blue G250 stain is reported, dubbed blue silver on account of its considerably higher sensitivity, approaching the one of conventional silver staining. SDS-PAGE gel staining with GelCode Coomassie Blue Stain Reagent saves time and cost while delivering superior results. Wash with water three times for 5 min each. However, its lengthy processing time is a major drawback of the sta- n dard Coomassie staining technique. It has a detection limit of 0.10.5 mg protein, sensitive enough for most daily needs. Coomassie blue protein stains can be used for sensitive, quantitative protein detection in gels, with a linear range from ~10 ng to 20 g. If you have to use Coomassie then colloidal coomassie staining is the only way you'll get high sensitivity and that's only down to around 5 ng protein per band. 825.99. Silver staining has greater sensitivity, but involves many more steps and solutions (see Silver Staining of SDSpolyacrylamide Gel). Supplied in a ready-to-use format of 1XStrength Solution, the staining solution is Fix gel in fixing solution (50:10:40 / methanol: acetic acid: H2O) for 25 - 30 mins. Features Easy-to-use: 1X ready-to-use form, fast stain in less than 20 min Coomassie R-250, the more commonly used of the two, can detect as little as 0.1 ug of protein. sensitive than silver staining, Coomassie Blue staining is a relatively simple and more quantitative method. Coomassie Sensible Blue staining resolution. Although traditionally used for densitometry, CBB has fluorescent properties. The ProtoGel Sample Prep Kit gives you the sharpest bands possible. Coomassie Brilliant Blue R-250 b- e cause of its relatively high sensitivity (3,6). Use freshly washed labware that has never been in contact with nonfat milk, BSA or any other protein blocking agent to prevent carryover contamination. Dark blotches on gel. Store at Room Temperature. A sensitive and convenient visible SYPRO staining protocol was developed for visualizing proteins after SDS-PAGE. Stain for 1 hour at room temperature with gentle shaking. Some premade and traditional homemade Coomassie R-250 protein stains can take three hours or more to fully stain gels, and then require destaining typically overnight. Coomassie G-250 manifests a leuco form below pH 2. Coomassie blue staining is a quick, simple, and affordable method for detecting proteins on gels. Uneven staining. Solubility overview. [2] (4) state that a staining time of at least 4 h is needed, with destaining overnight. Add 100ml of 85% H 3 PO 4 to the solution from step 1 3. Faint bands on a high background. colloidal coomassie blue (CCB) or SYPRO Ruby (SR), in addition to many others). You can also microwave it and watch the time. Procedure 1. First, regarding the colloidal Coomassie staining, there is a method dubbed blue silver by Giovanni Candiano published in the journal of Electrophoresis (2004) 25 1327-1333 that has a sensitivity in the 1 ng range and is less time consuming than you alluded to. Coomassie staining is a protein staining technique that uses a coomassie brilliant blue stain. It is generally called the coomassie blue technique. Coomassie brilliant blue is the most popular anionic protein-dye. This stain normally stains almost all proteins with good quantitative linearity at medium sensitivity. 3. A modified Neuhoff's colloidal Coomassie Blue G-250 stain is reported, dubbed "blue silver" on account of its considerably higher sensitivity, approaching the one of conventional silver staining. 10 fold higher than silver coloring, detecting barely 1 ng protein/mm2 gel. Coomassie stains (also known as Coomassie Blue or Coomassie Brilliant Blue) are the most popular anionic protein dyes used in visualizing proteins in SDS-PAGE gels. 1, 2. The main characteristics for an efficient stain are high sensitivity, low background, large linear range, and ease of use. There are several types of Coomassie Brilliant Blue staining solutions available, and protocols vary slightly, taking anywhere from two hours to a full day, depending on the vendor and solution type. 2. This method can be used for This stain is recommended for very low abundance proteins and is compatible with MALDI-TOF applications. A novel coomassie brilliant blue staining method, a related fixative and a related staining agent are disclosed. The eStain Protein Staining System combines three steps of traditional Coomassie blue staining method into one single step to greatly reduce the time required for protein staining (Table 1). A fluorescence-based Coomassie Blue protein staining. Fluorescent in-gel protein stains are generally preferred for higher sensitivity, reduced background, and wider dynamic range. Detection of the resulting protein maps relies on staining (i.e. Modified GelCode Blue Coomassie Stain Reagents 1. Staining with Coomassie Blue R250. Incubate tubes at room and vortex. It has a detection limit of ~ 0.10.5 g protein, sensitive enough for most daily needs. You can also microwave it and watch the time. Stain membrane with Coomassie blue stain Visible with the eye Great article Laura. Sensitivity is higher than Ponceau S (<10 ng of BSA in 10 mins as blue bands) and the staining is reversible in 5 minutes. Our stain can be used with SDS-PAGE, Native PAGE, Tricine gels with fixation step, 2-D Electrophoresis, etc. The most commonly used dyes in the laboratory for staining PAGE gels are Coomassie Blue R-250 and G-250. [1] Medical Uses Brilliant Blue G (BBG) has recently been used in scientific experiments to treat spinal injuries in laboratory rats. Disadvantages: complicated procedures and high cost. Stain cannot be re-used. Soluble in water to 10 mM. ProtoGel Quick-Cast: ready to run in 20 minutes! The unique patented mechanism for rapid Coomassie blue staining of protein gels begins in moments, and results are achieved within 15 minutes. combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. Blot transfer membrane (unit B3.2) Coomassie blue stain: 0.025% (w/v) Coomassie brilliant blue R-250 (Bio-Rad) in. 2. Dye that is not bound to protein diffuses out of the gel during destain steps. This capability of G-250 is due to its particular properties. This stain normally stains almost all proteins with good quantitative linearity at medium sensitivity. SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis) is a technique we use to separate proteins in a mixture by size (well, length) using electricity to send the proteins through a gel mesh. THEORY Coomassie blue staining is a quick, simple, and affordable method for detecting proteins on gels. The unique patented mechanism for rapid Coomassie blue staining of protein gels begins in moments, and results are achieved within 15 minutes. Coomassie brilliant blue stain binds non-specifically to almost all proteins. Though less sensitive, Coomassie G-250can be used in place of the R-250form to A modified Neuhoff's colloidal Coomassie Blue G-250 stain is reported, dubbed "blue silver" on account of its considerably higher sensitivity, approaching the one of conventional silver staining. While less sensitive as a colormetric method than silver, or fluorescent staining, Coomassie has undergone a significant revolution in recent years. Different Coomassie stains have different sensitivities (e.g. Coomassie R-250, the more commonly used of the two, can detect as little as 0.1 ug of protein. Destaining is not required, but may be performed to achieve maximum sensitivity, especially when performing downstream analysis such as mass spectrometry is required. Please sign in to view account pricing and product availability. Product Description: Coomassie Blue Stain Solution (Protein Stain-EZ A Ultra Sensitive) 1x Strength Solution: The Ultra-Sensitive Coomassie Blue Stain Solution, based on Coomassie dye (G-250), is an enhanced protein stain that offers unsurpassed sensitivity and rapid band visualization. (1985) Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysis. The SYPRO Ruby family of fluorescent dyes has high sensitivity, broad linear dynamic ranges and is compatible with mass spectrometry and other downstream protein identification (2,3).However, proteins stained by these fluorescent protein stains cannot be visualized by the naked eye. Add the solution from step 2 into 500ml of H2O and mix well 4. Why is Coomassie Blue a good stain for proteins? Save time and money by having us create a batch of 10X Tris-Glycine-SDS Buffer for you! The protocol involves soaking the gel in a dye solution. The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. Proteins stained using the Coomassie Blue Fast Stain are compatible with mass spectrometry (MS) analysis. Protein Stain-EZ A Ultra Sensitive Coomassie Blue Stain Solution: SK3011: Bio Basic: 10Minigel, 10UNIT: EUR 66.53: SafePinky DNA Gel Staining Solution(10,000X) In Water: S1001-025: GenDepot: Coomassie blue protein stains are anionic and bind non-specifically to proteins. Coomassie brilliant blue stain offers high sensitivity, low background, large linear range, and ease of use for the identification of proteins separated by gel. Sambrook, J., et.al. ProtoBlue Safe: Colloidal Coomassie Stain. Bio-Safe Coomassie (161-0786) Bio-Safe Coomassie Brilliant Blue G-250 stain is fast, simple, sensitive, and convenient. Gel-Code Blue stain Reagent (PIERCE Cat. The most widely used protein stains are Coomassie Brilliant Blue R-250, Coomassie Brilliant Blue G-250 (colloidal Coomassie), silver staining, and the fluorescent dye SYPRO Ruby. There are several reasons: Low price Visible with the eye Silver staining: Sensitivity: 2ng/band. Premixed, ready-to-use, nonhazardous solution No methanol or acetic acid required for destaining Bio-Safe composition reduces solvent waste disposal costs The gels in Figure 3 demonstrate the superior staining results achieved with SimplyBlue over other Coomassie stains. 40% methanol/7% acetic acid (v/v) 50% methanol/7% acetic acid (v/v) Plastic box. so proteins remain blue. There are two different coomassie brilliant blue stain variants: R-250 and G-250. Stain the mini-gel with enough Invitrogen SimplyBlue SafeStain (20-100 ml) to cover the gel. Thermo Scientific GelCode Blue Stain Reagent is a ready-to-use protein stain based on colloidal coomassie dye G-250 that provides nanogram-level detection and results that exceed the clarity obtained with typical coomassie stains. Silver staining: Sensitivity: 2ng/band. 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 Coomassie brilliant blue stain offers high sensitivity, low background, large linear range, Coomassie Blue stain is used to stain the protein bands in polyacrylamide gels. (4) state that a staining time of at least 4 h is needed, with destaining overnight. Dark blotches on gel. However, its sensitivity depends on the type of stain (Coomassie Brilliant Blue G-250 and Coomassie Brilliant blue R-250) used in the staining method. Stain solution composition: 5% Coomassie Blue G250 Stain solution preparation: 1. I just wanted to pitch-in a little bit more information here and there. HPLC water or Mill-Q water. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots Find out more about our easy-to-use coomassie stains for protein gels and membranes for visualization of proteins resolved by protein gel electrophoresis (SDS-page). Coomassie blue protein gel staining began in the 1960s, and its still a fan favorite in biology labs today. Bio-Safe Coomassie Stain is a nonhazardous formulation of Coomassie Blue G-250 that requires only water for rinsing and destaining. Although G-250 is more sensitive, R-250 affords better resolution, and is often used instead. Procedure for Staining Membranes Chu, R. and Vigna, R.A. (1998).