The protocol includes only staining and quick washing steps, whereas traditional post-electrophoretic process is time consuming and cumbersome because it involves fixing, staining of protein gels, and destaining, and requires several hours to several days to finish the whole staining process [19][21]. The user manually cryptically states InstantBlue contains Coomassie [sic] dye, ethanol, phosphoric acid and solubilizing agents in water. DISCLAIMER: ConductScience and affiliate products are NOT designed for human consumption, testing, or clinical utilization. broad scope, and wide readership a perfect fit for your research every time. Zinc ions coupled with imidazole precipitates in the gel matrix. The epicocconone stain is a fluorescent stain that was isolated from the fungus Epicoccum nigrum. PLOS ONE promises fair, rigorous peer review, One disadvantage for fluorescent stains is they are generally more expensive than colorimetric methods and require a fluorescence imager. In the present study, we present an improved method for in-gel staining of proteins, which has the advantage of speed over the conventional CBB staining, yet has a comparable sensitivity. It wouldnt be a great article if it wasnt information-dense! Mix-n-Stain CF Dye Antibody Labeling Kits, TrueBlack Lipofuscin Autofluorescence Quencher, Scaling Down to Go Big: An Overview of the Lab-on-a-Chip, Optical Imaging in Tissue with Near-Infrared Dyes, Bring Out Your Dead: v-PCR for Microbial Population and Diversity Studies, Receive 20% Off Your Next Order by Subscribing to Our E-Newsletter, Biotium implements a Quality System, certified by QAS according to Standard QAS ISO 9001:2015. Advantages: Convenient, sensitive, wide linear range and compatible with multiple downstream analysis. The color of the bands and backgrounds became more unclear as the staining time increased. Use for: Gel stain only when you need sub-nanogram sensitivity for total protein. Moreover, silver staining presents worse mass-spectrometry (MS) compatibility compared with the traditional Coomassie blue staining because it includes glutaraldehyde in the sensitization solution. Acquire the images with UV or blue or green visible light. Many protocols for protein staining using colloidal coomassie are available, several of which are described and compared in this paper by Dyballa and Metzger. Protein samples were loaded into the individual wells, and electrophoresis processed 1 mA/cm gel in the stacking gels and 2 mA/cm gel in the separating gels. The increased sensitivity of this protein staining method over classical coomassie makes it great for experiments involving low protein levels, such as analyzing binding proteins in co-IPs, especially when youre considering quantifying the protein bands using mass spectrometry. The entire method takes 15 minutes. No, Is the Subject Area "Staining" applicable to this article? These conveniences allow Ponceau to be a quick method for visualizing total protein on western blots. He is the current Marketing Manager at Biotium. Yes Originally written and published September 2012 by Laura Grassie. Has this article on protein staining methods been a boon? High-fluorescent background and photostability are the limitations of this staining technique. 3). Bovine serum albumin (BSA) and CBB R-250 were purchased from Takara Co., Ltd., Japan. 4). When Im purifying proteins for X-ray crystallography, I dont have the time to wait for my gels to destain before I can be sure Ive actually expressed my target. R. Therefore, we suggest boiling the solution at a lower temperature to avoid breakage of the sheet. Polyacrylamide gel electrophoresis (PAGE) has become the most common method for protein analysis and detection in molecular biology experiments [1][4]. And also you can do chloroform staining prior to coomasie staining if you have tryptophan residues in your protein, as chloroform binds to tryptophan and fluoresces under UV light. https://doi.org/10.1371/journal.pone.0022394.g001. For more information about PLOS Subject Areas, click Coomassie brilliant blue stain is easily available and is the most commonly used protein stain. After separation, fix the gel in fixation solution (50% methanol, 12% acetic acid, and 0.05% formalin) for 2 hours or overnight, followed by three washes with 35% ethanol for 20 minutes each. A summary of silver, coomassie, colloidal coomassie, and InstantBlue stains. Never waste antibody with these optimized kits for labeling phycobiliproteins. SYPRO Ruby is a luminescent ruthenium metal chelate that interacts with the basic amino acids in proteins. Silver staining is my least favorite of the protein staining methods, not just because of the multiple steps required but also due to the use of several dangerous chemicals (including silver nitrate and formaldehyde), which I like to avoid if possible. This protein staining method is my personal favorite for simple tasks such as visualization of recombinant proteins, or generated antibodies, as its simple and quick to perform. Or simply cant be bothered with all that staining and destaining malarkey? The zinc stain is a sensitive dye for the detection of 0.25 ng of protein per band in a mini gel. Protein separation and identification is critical for proteome analysis and requires high resolution and powerful protein characterization after gel electrophoresis. Click through the PLOS taxonomy to find articles in your field. After separation, the gels were carefully transferred to a steel tray filled with distilled water, heated to 100C, and cooled to room temperature to remove the water. No, Is the Subject Area "Distillation" applicable to this article? No, Is the Subject Area "Boiling" applicable to this article? The gels can be viewed under UV light or with green light sources; the protein bands appear pale red. All reagents used were of analytical grade. After electrophoresis, incubate the gel in 0.2 M imidazole, 0.1% Sodium dodecyl sulfate SDS for 15 minutes. Fancy a game of spot the difference? We found that water has slight effect on the staining results (Fig. In fact, several studies have found that normalization by total protein is superior to normalizing with housekeeping proteins such as tubulin or actin.13Yet each protein stain is unique, with inherent advantages and disadvantages that should be considered when interpreting results. It also allows a stable sequence coverage with a capacity for spots identification. The mechanism for hot fast CBB staining with the rise of temperature is possibly because of the increasing speed of the molecules of the dye and proteins. In conclusion, the protocol described in the present study has four advantages: simplicity, low cost, environmental friendliness, and time-saving ability. Competing interests: The authors have declared that no competing interests exist. Therefore, proper animal identification becomes a necessity. Prior to electrophoresis, the samples were heated in the presence of sample buffer (70 mM TrisHCl, pH 6.8, 11.4% glycerol, 3% SDS, 0.01% bromphenol blue, and 5% b-mercaptoethanol) at 100C for 5 min in a boiling water bath [15]. Funding: This work was supported by the National Natural Science Foundation (30970055/C010202), http://159.226.244.28/portal/Proj_List.asp. Subsequently, a protocol for staining proteins in polyacrylamide gels using a methanol/acetic acid/water mixture (515) as solvent system for CBB R-250 was described [9][12]. Additionally, van der Waals forces and hydrogen bonding contribute to the binding interaction between dye and protein [18]. The structures of G-250 and R-250 are shown in Figure 1 below. However, staining and destaining require more time and reagents. In spite of the high sensitivity of silver staining and the wide dynamic range of various fluorescent detection methods, Coomassie Brilliant Blue (CBB) staining remains the most commonly used detection technique for proteins separated by electrophoresis [5], [7]. In a rush? For more information about PLOS Subject Areas, click Increasing the purity or lengthening the destaining time from 10 to 12 h can produce a clearer background. Two main types of coomassie staining exist: Need a reliable, bread and butter protein staining method that you can share with your lab group? Its another coomassie-based protein stain but formulated in such a way to provide one-step visualization of your proteins. These reagents are not only expensive but also cause environmental pollution. Important considerations to help you obtain the best possible data for your next v-PCR experiment. You just have to soak the gel in chloroform for 1 min wash it MQ water until the chloroform is removed and then view it under UV light. CBB staining was first developed to stain proteins on a cellulose acetate sheet in 1963 [8]. The VersaBlot Total Protein Normalization Kit comes with your choice of two near-IR CF Dyes for emission in Odyssey 700 or 800 channels. To evaluate the sensitivity of this approach, different concentrations of BSA samples were separated on SDS-PAGE. It provides a sensitivity close to that of silver staining and the properties of classical organic stains such as Coomassie blue. PLoS ONE 6(8): There was no significant difference between washing with distilled water and washing with double-distilled water. VersaBlot, Convenient, Highly-Linear and Reversible. https://doi.org/10.1371/journal.pone.0022394.g003. Therefore, boiling temperature was necessary to obtain a clear band and clean background. silver stain, fluorescence) and has a narrow linear range. We compared the gel staining under different boiling times and different staining times at room temperature. Use for: A convenient method for western blot normalization and downstream multi-color near-IR protein detection. You can re-use it, but siphon off the re-used stuff into a separate opaque container and discard it when you notice its no longer staining your proteins. It is highly compatible with mass spectrometry, Edman sequencing, and Ettan DIGE system. The work is made available under the Creative Commons CC0 public domain dedication. In honor of our newly released protein prestain, VersaBlot, we decided to give our readers a guide for the some of the most common protein stains which includes a close look at their origins, advantages and limitations. InstantBlue protein stain can detect proteins down to ~ 5 ng. However, staining with this quick staining solution at room temperature was difficult, and no clear bands were acquired even after staining for 24 h (Fig. This creates a visible image. Yes The low reproducibility of classical coomassie staining is also a disadvantage due to the difficulty in standardizing the destaining step. Zinc ion staining utilizes the ability of proteins and protein-SDS complexes to bind and sequester Zn2+ in the gel, causing the precipitation with opaque background, contrasting with transparent protein-SDS-Zn2+ zones. Filer the solution with Whatman #1 paper for a stable formulation. If you plan on western blotting, stain the membrane after transfer and before blocking. So it can be used as initial screening of your protein bands. The classical coomassie protein staining technique involves incubating the gels with a coomassie staining solution. The stain could be profitably used for large-scale proteomic analysis. Boiling for 3060 s was found to be enough to obtain a clear band (Fig. You didnt touch on Ponceau S staining which I really like for checking complete transfer of WB, and to a lesser extent to detect equal loading of protein samples. Washing before staining is necessary to prepare a clean background. Analyzed the data: FW. Is the Subject Area "Coomassie Blue staining" applicable to this article? Colloidal coomassie has several advantages over classical coomassie protein staining. At present, SYPRO orange, SYPRO red, Deep Purple, and Flamingo are frequently used in proteomics research. 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. Subsequent to separation by electrophoresis, proteins in a gel are detected by several staining methods, such as Coomassie blue, silver, and fluorescent staining methods. However, fluorescent staining is inconvenient because it requires special equipment, such as a fluorescent imaging scanner and an integrator [5][7]. Silver staining can detect ~ 1 ng of protein [1] which makes it extremely useful for applications involving low protein levels. This indicates better time-saving ability compared with conventional Coomassie [16] and other fast Coomassie staining methods [15], [17]. His dissertation research focused primarily on mechanisms of mitochondrial protein import. In the dye stain, no acids are needed for the staining solution preparation. Given that no destaining is required, it must act similarly to colloidal coomassie and stain specifically protein molecules and not the gel. Use for: Gel or membrane when you need an inexpensive total protein stain. No, Is the Subject Area "Silver staining" applicable to this article? 4, lane 1). Sign up for our feature-packed newsletter today to ensure you get the latest expert help and advice to level up your lab work. 1). What Does Vaping Vitamin E Acetate Do To Your Lungs? As protein biologists, these stains play a crucial role in our day-to-day lab work for routine protein analysis and western blot normalization. Moreover, the staining is irreversible and for gel use only and therefore not suited for downstream analysis such as mass spectrometry or western blotting. Despite these advantages, CBB lacks sensitivity relative to alternative staining methods (ie. Silver staining is considered the gold standard for sensitive protein detection methods. Coomassie brilliant blue stain is a disulfonated triphenylmethane dye that is used to stain protein bands bright blue. Discard the imidazole solution and incubate the gel in 0.3 M zinc sulfate for 30-45 seconds. Youll stay up-to-date with our podcasts, webinars, workshops, downloadables, and more, delivered to your inbox every fortnight. CBB was first used as an acid wool dye in the 19th century by a British dye manufacturer named Levinstein Ltd. Fast-forward to 1964, an Australian biochemist named Fazekas De St. Growth and colleagues discovered its application for staining proteins after gel electrophoresis.4Since then, several versions of CBB have been developed, including the widely accepted colloidal G-250 formulation thats more sensitive and has less background than the older R-250 form. Silver staining is one of the most sensitive protein-staining methods and could detect proteins at nanogram levels. All good things come at a cost, however, and colloidal coomassie kits will cost you a bit more than your regular coomassie stain. Stain the gel in silver nitrate solution (0.2% silver nitrate and 0.076% formalin) for 20 minutes, followed by two washes in water. Learn how your comment data is processed. It is an azaphilone that reacts with primary amines and NH3to produce red fluorescent compounds which detect proteins in the gels. Youll see your protein bands in 1015 minutes. Eric Torres holds a PhD in Biochemistry and Molecular Biology from the University of California, Los Angeles. InstantBlue protein stain works fast. It is generally considered as the standard for other ultrasensitive staining methods. The gel images were acquired by Tocan 240 system (Tocan Biotechnology Corporation, Shanghai, China) in a UV-1 mode. Nothing left to fear about labeling your antibody with PE, APC, and PerCP. Discover a faster, simpler path to publishing in a high-quality journal. here. e22394. The staining method for protein identification in gel should be chosen based on its sensitivity and downstream analysis techniques. Silver staining also has a narrow linear dynamic range (the range at which the level of staining is directly proportional to protein concentration), making it less suitable for quantification. The staining method for protein identification in gel should be chosen based on its sensitivity and downstream analysis techniques. Fluorescent staining could detect proteins at the nanogram level without the requirement of special technical skill. CBB R-250 (500 mg) was dissolved in 1000 ml of distilled water by stirring for 24 h. Subsequently, the solution was heated to 50C for complete dissolution of CBB R-250. Disadvantages: More expensive than colorimetric methods, requires a near-IR fluorescence imager. Yes The protein stain should exhibit high sensitivity (i.e., low detection limit), quantitative accuracy, reproducibility, ease of use, and compatibility with downstream protein analysis techniques like mass spectrometry (MS). The backgrounds were very difficult to retain (Fig. However, it has the following disadvantages: difficulty in the operation and low repetition rate. https://doi.org/10.1371/journal.pone.0022394, Editor: Vladimir N. Uversky, University of South Florida, United States of America, Received: May 18, 2011; Accepted: June 20, 2011; Published: August 5, 2011. Nile red does not bind the SDS monomers; this property makes it a rapid, non-fixative total protein staining method for SDS gels. This article explains the pros and cons of three of the most common protein staining methods: silver staining, coomassie brilliant blue staining, and InstantBlue staining. If youre struggling to see faint protein bands, soak your developed gel in water to increase their contrast. However, as the research community gradually shifts toward fluorescence-based technologies, fluorescence imagers are becoming far more common and affordable. Affiliation The classical coomassie protein staining method is cheap and easy, but is considerably less sensitive than silver staining, making visualization of low abundance proteins difficult. The technique was first developed for SDS-PAGE by Switzer et. Because the colloidal dye does not penetrate the gel, no destaining is required. 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. While selecting the stain, the composition of the proteins of interest (presence/ absence of single amino acids), sample availability, and post-staining application of the gel/protein should be considered. Concentrations of BSA were determined by the Bradford method [15]. Sundaram., N. The silver cations bind to the target protein(s) and are subsequently reduced to silver metal using a developing solution. When the gels were washed using tap water, the bands were clear, but the sensitivity was worse than those of the bands washed with distilled water and double-distilled water (Fig. Advantages: Cheap with excellent sensitivity. It is also environmentally friendly and does not contain any heavy metals, allowing safe disposal after use. But how do you know which protein staining method to use? Disadvantages: Poor sensitivity and reproducibility. Have we missed some salient facts out? A guide for protein staining should rightfully begin with the most widely used protein stain, Coomassie Brilliant Blue (CBB). Our method does not require the use of any acid and methanol nor tricarboxylic acid. 3, lane 7). The results showed that this approach has a sensitivity of 10 ng, which is the same as the traditional Coomassie blue stains [16] and other fast Coomassie stains [15], [17] (Fig. Thats why I use the InstantBlue protein staining method. Incubate the gel in 0.05% (v/v) ammonia (310 min). No, Is the Subject Area "Water pollution" applicable to this article? Silver staining is one of the most sensitive colorimetric methods used for the detection of total protein. No, PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US, Corrections, Expressions of Concern, and Retractions, https://doi.org/10.1371/journal.pone.0022394, http://159.226.244.28/portal/Proj_List.asp. However, it still requires a long staining time, and the relatively complicated ingredients make its use inconvenient. And like the other coomassie-based stains its fully compatible with mass spectrometry. The technique is compatible with mass spectrometry or microsequencing methods for downstream protein analysis and characterization. To evaluate whether the acids have any effects on the protocol, we used different acids, including hydrochloric acid, phosphoric acid, and acetic acid, to prepare the staining solution in the experiment. Classical coomassie has a lower limit of detection of ~ 100 ng. Perform the fixation and staining in 10 gel volumes of solution. Can be used for several applications including western blot normalization and mass spectrometry. Yes Proteins could be easily recovered from the gel after staining. This process is called destaining. Learn more about our new VersaBlot Total Protein Normalization Kits and our other products for protein detection and analysis. Biotium has furthered the advancement of fluorescent protein detection with VersaBlot, a highly linear, sensitive and reversible prestain. 5250 Old Orchard Rd Suite 300Skokie, IL 60077, This website uses cookies to improve your experience. Staining proteins following SDS-PAGE or 2-dimensional electrophoresis is necessary for visualizing a global population of proteins or checking the expression of your recombinant proteins. This stains the entire gel, not just the proteins. The stain binds with the protonated basic amino acids (lysine, arginine, and histidine) by electrostatic interactions and with the aromatic residues by hydrophobic interactions. Yes, what Min has said about microwaving your gel is right. Switching from agar plates to v-PCR for bacteria viability testing What you need to know. BSA (500 ng) was separated by SDS-PAGE and stained with different acid-soluble CBBR at boiling temperature for 60 s. Added to the water-soluble CBBR staining solution were 0.3% HCl (Lane 1), 0.3% phosphoric acid (Lane 2), and 0.3% acetic acid (Lane 4), respectively. Coomassie stains are noncovalent, reversible, and do not interfere with downstream analysis techniques such as mass spectrometry of excised protein bands. Fix the gel in 50% (v/v) methanol, 10% (v/v) acetic acid, and leave it for 30 minutes or overnight. Yet, unlike CBBs longer staining protocol (at least 45 minutes plus destaining) Ponceau staining can be done in a few minutes and can be quickly destained with water. Standard Dyes for Total Protein Staining in Gel-Based Proteomic Analysis. The staining does not lead to any irreversible modification of amino acids, so satisfactory mass spectrometry compatibility is expected. Acrylamide and bis-acrylamide were purchased from Sigma (St. Louis, MO, USA). With this method, I get perfectly stained gels in just 4 hrs (3 x 10 min for washing gels in MQ water, 3 h staining with the blue silver, 30 min destaining with MQ water). What we know as Ponceau in our labs is the Ponceau S formulation, a reversible total protein stain for western blot membranes. [2]. Click here As an Amazon Associate Conductscience Inc earns revenue from qualifying purchases The modern pipette has had a colorful history, Scientific investigations, preclinical research, and pharmacological studies use a number of laboratory animals as subjects. This method is an adaptation of classical coomassie staining that uses a modified coomassie dye (G-250 instead of R-250). Here is a helpful table summarizing the protein stains discussed in this article and stains offered by Biotium. The stain is a rapid and sensitive fluorescent total protein stain suitable for both protein gels and blots. Performed the experiments: WHD. Inverted Light Microscope: A Comprehensive Guide for Students of Microbiology and Laboratory Technicians. Add a tenfold volume excess (e.g., 50 mL staining solution for a 5 mL mini gel) to a gel and thoroughly agitate. Add the developer (6% sodium carbonate, 0.05% formalin, and 0.0004% sodium thiosulfate) and stop staining by leaving the gel for 5 minutes in the stop solution (50% methanol and 12% acetic acid). US3564. 3). Hopefully, youre now armed with enough information to choose the protein staining method thats most appropriate for your intended experiments. Zinc staining is a negative stain that stains the polyacrylamide gel except proteins enabling their detection. Id love to be able to tell you how this protein staining method works, but that big registered trademark symbol means theres a bit of an information vacuum. Unlike colorimetric or fluorescent protein stains where staining is done after running your gel, VersaBlot is added to your sample before electrophoresis and can be used for downstream western blot normalization. Specific staining methods could also be used to detect post-translational modifications such as glycosylation or phosphorylation. Silver staining protocols do exist that are compatible with mass spectrometry but at the sacrifice of sensitivity. To identify the proteins in the gel, colorimetric and fluorescence staining techniques are used. The running buffer was prepared from 25 mM Tris base, 0.2 M glycine, and 0.1% sodium dodecyl sulfate (SDS) with pH of 8.3. Great article Laura. Discard the developer and wash the gel for several times, with water, 1 minute per wash. Store the gel in 0.5% (w/v) sodium carbonate. A microscope is an essential tool that is used in most laboratories. Different concentrations of BSA were separated by SDS-PAGE and stained with water-soluble CBBR at boiling temperature for 60 s. Lanes 16 represent 10 ng, 100 ng, 500 ng, 1 g, 5 g, and 10 g of BSA. It helps you save a lot of time but you have to be careful so that you dont overheat the gel or it might dehydrate, other than that it has no detrimental effect on the gel. The water solution with and without acid could dye the protein just as well in this staining protocol (Fig. Silver diamine complex in an alkaline environment is used in alkaline methods; acidic silver nitrate is used in acidic methods. Disadvantages: Lacks sensitivity, small linear range, dye may interfere with downstream antibody binding and mass spectrometry analysis. Ponceau is one of the least sensitive staining techniques with a detection minimum of 200 ng.5In addition, the stain is considerably weaker than CBB and may fade overtime, making normalization less reproducible between experiments. The addition of 10% acetic acid to the destaining solution can also help in achieving better destaining effects. Yes In addition, the staining protocol requires only a single prepared reagent and binds to proteins non-covalently through interactions with basic and aromatic residues. Thank goodness for InstantBlue. Wrote the paper: TYW WHD. Dissolve 0.0250.10% (w/v dye) dye in an acidic alcohol formulation [30 50% (v/v) methanol (or, less frequently, ethanol)) with 710% (v/v) acetic acid solution. Colloidal coomassie has a detection limit of ~ 10 ng, making it about an order of magnitude more sensitive than classical coomassie. [16]. Nile red is a phenoxazone dye that presents strong fluorescence upon transition from aqueous to hydrophobic environments like SDS micelles or proteinSDS complexes. Stain the gel for 1 hour in an aqueous solution into which an aliquot of the fluorophore stock solution is diluted. 2, lane 3).