Stain-free imaging technology utilizes a proprietary polyacrylamide gel chemistry to make proteins fluorescent directly in the gel with a short photoactivation, allowing the immediate visualization of proteins at any point during electrophoresis and blotting.
Because the fluorophores are covalently bound to the protein molecules, they can be imaged repeatedly on the gel or on a membrane after transfer, without staining and destaining steps. Protein electrophoretic mobility, transfer, and immunodetection properties are unaltered.
Furthermore, stain-free imaging allows for the elimination of the inherently problematic use of housekeeping proteins as loading controls on western blots, permitting the user to obtain truly quantitative western blot data by normalizing bands to total protein in each lane.
Polyacrylamide gel electrophoresis (PAGE) is a well-established method for the separation, detection, and analysis of proteins in the first step of western blotting analyses. After separation, proteins are visualized on polyacrylamide gels using protein stains such as Coomassie Brilliant Blue (CBB). Other stains, such as silver, zinc or some fluorescent dye-based stains are also used to determine sample quality, level of separation, and protein load. These traditional staining methods are generally time consuming, require fixing the protein in the gel, and preclude the use of gels in downstream applications, such as western blotting and mass spectrometry.
Stain-free technology addresses these issues and has changed the protein separation and analysis landscape in the last few years. Stain-free technology has been adopted by many labs for its simplicity, ease of use, and, most important, its reliability, which confers validity to the results obtained in downstream applications. Gels developed with stain-free technology do not require the use of specialized buffers or reagents and are compatible with standard SDS-PAGE buffers. Imagers enabled with stain-free detection capabilities can visualize activated gels or membranes, allowing protein detection in a gel both before and after transfer, as well as total protein detection on a blot using wet nitrocellulose and low-fluorescence PVDF membranes.
Stain-free technology utilizes a proprietary trihalo compound to enhance the fluorescence of tryptophan amino acids when exposed to UV light. A 58 Da moiety is covalently bound to the tryptophan, enabling the detection of proteins down to 20–50 ng. The addition of the moiety does not interfere with downstream steps and allows detection of the proteins in the gel, as well as following transfer during Western blotting.
Commercial stain-free technology utilizes proprietary trihalo compounds contained in stain-free gels, such as Bio-Rad's Mini PROTEAN® TGX Stain-Free™ Gels and Criterion™ TGX Stain-Free™ Gels, to covalently bind to tryptophan residues in proteins when activated with UV light. Certain aromatic amino acid residues, such as tryptophan and tyrosine, undergo specific chemical reactions and form key intermediates, such as kynurenine, cyclic lactams, and Dopa, when exposed to UV radiation. Compared to UV activation alone, adding the trihalo compound enhances the rate at which the chemical reaction produces fluorescence. Activation of the trihalo compounds in the gels adds a 58 dalton moiety to available tryptophan residues and is required for protein visualization. The resulting tryptophan adducts emit fluorescence upon excitation by another brief irradiation from stain-free enabled imagers, facilitating visualization and quantitation of proteins from gels, as well as from blots after the proteins are transferred or probed with antibodies for western blotting. Proteins that do not contain tryptophan residues are not detected; however, in commonly studied organisms, 90% of proteins contain at least one tryptophan and the majority of proteins lacking tryptophans are less than 10 kD in size (Table 1).
With the stain-free method, modifications to the proteins themselves are minimal and do not affect protein transfer or downstream antibody binding in western blotting, nor does the binding mimic any posttranslational modifications, which could potentially result in misinterpretation of results. Also, unlike CBB-stained gels, stain-free gels can be used for downstream applications such as western blotting (Elbaggari et al. 2008; Gilda and Gomes 2013) or mass spectrometry (Liu et al. 2008, Susnea et al. 2013, Gonzalez-Fernandez et al. 2013), making stain-free technology superior to most dye-based visualization techniques. The limit of detection for the visualization of proteins rendered by this method is 20–50 ng of protein compared to the 100 ng range of the standard CBB staining method or the 8–15 ng range of colloidal CBB (Ladner et al. 2006).
Stain-free gels incorporate the trihalo compound in their gel formulation and are run with standard protocols and reagents like any other gel used in SDS-PAGE. Unlike with Coomassie or other dyes, there is no destaining step, and stain-free technology is environmentally safe and does not generate toxic or hazardous organic waste.
Visualization of Proteins on Gels – Stain-Free Technology Provides More Sensitivity and Better Dynamic Range than Coomassie Stains
Protein visualization data obtained from stain-free gels are comparable to those obtained from gels stained with other dyes. In general, the sensitivity of stain-free gels when visualizing data is equal to that of Coomassie-stained gels for all proteins. For proteins with higher tryptophan content, stain-free gels provide much higher sensitivity than CBB-stained gels (Figure 1).
Figure 1. Comparison of a stain-free gel image and CBB R-250−and Bio-Safe G-250−stained gel images. Serial 1:2 dilutions of broad range unstained molecular weight standards were separated on a 4–20% Criterion Stain Free™ Tris-HCI Gel. The gel was imaged with a stain-free enabled imager, then stained with Coomassie (CBB R-250 and Bio-Safe G-250) stain and imaged on a densitometer. Arrowhead indicates β-galactosidase.
The limit of detection for the stain-free gels is 8 to 28 ng, similar to that of silver stains (0.6 to 1.2 ng), while Coomassie R-250 stain can detect protein amounts of at least 35 to 50 ng. Some fluorescent stains can detect proteins at levels below the 1 or 0.5 ng limit. Stain-free gels have more reproducible data with smaller coefficients of variation compared to Coomassie or silver stains and allow the quantitation of 0.2 ng of proteins (McDonald et al. 2008; McDonald, 2009).
The linear dynamic range for protein quantitation is defined as the range through which the signal intensity on a blot proportionally increases with the increase in protein load. Ideally, the protein load should fall within the quantitative linear dynamic range of the antibody used for its detection (Taylor and Posch, 2014). Stain-free gels provide a linear dynamic range between 10 and 80 µg of total protein load from cell or tissue lysates at a higher range of protein load (Figure 2A) and from 20 to 1 µg at a lower range (Figure 2B) (Taylor et al. 2013, Hammond et al. 2013).
Figure 2. Linear dynamic range provided by stain-free technology for total protein measurements. A, HeLa cell lysate dilutions from 80–2.5 µg total protein; B, HeLa cell lysate dilutions from 20–1 µg total protein.
Compatibility with Downstream Applications
The Coomassie dye-based method does not allow the same gel to be used for transfer during western blotting or for mass spectrometry. This is a serious drawback when the ultimate goal is the identification or quantitation of proteins in western blotting, especially when large gels and sample volumes are required for separation and visualization. In contrast to Coomassie staining, stain-free technology is compatible with most downstream applications, such as western blotting, which helps not only when resources are limited, but also in the appropriate normalization of data for quantitation using total protein normalization (see Normalization of Data in Quantitative Western Blotting below).
Verification of Protein Transfer
The proper transfer of proteins to the membrane is critical to the western blotting process. A common standard practice to verify protein transfer is staining the blot with Ponceau S, a negatively charged stain that binds to all positively charged amino acids in a protein. Ponceau S staining is fast and relatively inexpensive, and the stained membrane can then be used for downstream applications such as western blotting. However, the ephemeral nature of the binding of the dye to the protein causes the intensity of the bands on the membrane to decrease rapidly, making detection and quantitation harder. Other relatively expensive blot stains, such as SYPRO Ruby or amido black, are also used for blot quantitation and require special disposal methods. The time needed to view the stain on the blot also varies with the dye used. Protocols for using Ponceau stain recommend at least 5 minutes of staining followed by 15 minutes of washing steps. SYPRO Ruby Blot Stain is more elaborate, requiring fixation, overnight staining, and destaining.
Verifying protein transfer, both from the gel and on the membrane, using a stain-free enabled imager provides a significant time and cost savings for western blotting (Colella et al. 2012). The observed intensity of the bands does not depend on the duration of staining or destaining, a factor affecting dye-based techniques during visualization and quantitation. Also, the intensity of the band on stain-free blots, which is the result of a covalent modification, does not decrease with time. Using the stain-free method, protein transfer can be verified in as little as 2 minutes (Figure 3).
Figure 3. Assessment of protein transfer using a stain-free enabled imaging system. Images of the gel, before and after transfer, and of the membrane after transfer were taken using a stain-free enabled imager. Serial 1:2 dilutions of hemoglobin (starting quantity, 80 ng), with 1.8 µg of BSA/lane as a carrier (top band), were electrophoretically separated on a 4–20% 26-well Criterion Stain Free Gel.
As stain-free technology utilizes the modification of tryptophan residues, the primary concern is whether your protein of interest contains tryptophan residues. Proteins that lack tryptophan residues, such as aprotinin, are not detected using this technology (Figure 4). A single tryptophan residue is sufficient for signal activation and proteins that have as few as two tryptophan residues are readily detected and quantified using stain-free technology.
Figure. 4. Limits of detection (LOD) and limits of quantitation (LOQ) of proteins on Criterion Stain Free– and Bio-Safe G-250–stained gels. Individual bands from broad range unstained protein standards from four replicate gels were used to determine visual LOD and LOQ. Averaged numbers were used to generate the graph.
Data available from UniProt show that only about 10% of proteins from all commonly studied organisms lack tryptophans and that most of those proteins are less than 10 kD in size. In most organisms, approximately 90% of proteins are above 10 kD in size (in the 10–260 kD range) and possess tryptophan residues (Table 1).
Table 1. Tryptophan content of the predicted proteomes of several model organisms.*
* Sequence data were obtained from the UniProt database.
A computational study investigating the frequency of individual amino acids in the nuclear, cytoplasmic, and integral membrane proteins in the SWISS-PROT database showed that tryptophan is present in all these fractions (Schwartz et al. 2001). Even though tryptophan is generally found less frequently than other amino acids (Schwartz et al. 2001), most proteins can be detected and quantified using stain-free technology with greater sensitivity and lower detection limits than with Coomassie staining (see Figure 1).
Another common concern when considering a new technology is how this technology will impact existing workflows and necessitate the use of specialized reagents and consumables. Stain-free gels do not use specialized buffers or reagents. Standard SDS-PAGE buffers can be used. However, the technique does require an imager that can visualize the activated gel or membrane.
Western blotting is widely used as a semiquantitative method for the measurement of protein expression. Changes in expression levels are determined by comparing band intensities between different samples or different experimental conditions. To be reliable, band intensity obtained from the target protein needs to be normalized against a reference whose intensity should only vary proportionally to the quantity of material in the sample. Housekeeping proteins, such as actin, β-tubulin, or GAPDH, whose expression is not expected to change with experimental conditions or between different tissue samples, are often used for target protein normalization. However, recent findings suggest that the expression of these commonly used control proteins is not constant across different experimental conditions, tissues, or genetic backgrounds (Pérez-Pérez et al. 2012; Li and Shen 2013; Dittmer and Dittmer 2006; Rocha-Martins et al. 2012; Kosir et al. 2010) (see Figure 5). In addition, these highly expressed proteins tend to yield oversaturated bands in western blotting that can make quantitation difficult.
Stain-free technology eliminates many of the issues related to signal normalization for accurate protein quantitation. Normalization is performed by measuring total protein directly on the membrane that is used for western blotting. The total density for each lane is measured from the blot and a lane profile is obtained. Specialized software from stain-free enabled imagers can interpret the data from the lanes in three dimensions, so the same lane profile data can be viewed as a three-dimensional peak. The background is adjusted in such a way that the total background is subtracted from the sum of density of all the bands in each lane (referred to as the rolling disk background subtraction algorithm).
Figure 5. Comparison of protein normalization using stain-free technology and commonly used housekeeping proteins. Tenfold dilutions of HeLa cell lysates ranging from 50 to 10 µg were loaded for samples detected with stain-free technology (A) and the housekeeping genes β-actin (B), β-tubulin (C), and GAPDH (D). The protein quantification signal is higher with stain-free technology than with housekeeping genes (E).
Total protein normalization eliminates the need to strip and reprobe blots for housekeeping proteins to normalize protein levels. Therefore, the method saves time and improves the precision and reliability of Western blotting data. Total protein normalization using stain-free technology has also been shown to be more effective at enabling the detection of smallfold differences in protein expression and regulation compared to normalization using housekeeping proteins (Gurtler et al. 2013).
In terms of relative intensity, the signal intensity produced from stain-free gels is much stronger than that from Ponceau-stained membranes. Stain-free technology provides a superior linear dynamic range compared to that using Ponceau S total protein staining (Gilda and Gomes 2013).
Total protein normalization also circumvents issues related to the stripping and reprobing of the membranes, such as raising the antibody for housekeeping proteins and proteins of interest in the same organism. Although stripping and reprobing is a commonly used procedure, it is important to ensure that the primary antibody previously used is removed completely and that there is no residual signal from that particular antiserum after the membranes are stripped. This essential verification step, which is often skipped, requires reincubation of the blot in the ECL substrate and exposure to film before the process of reprobing begins (MacPhee 2010). Because stain-free technology does not require immunodetection of a housekeeping protein for normalization, reprobing steps can be avoided altogether.
With an increasing number of errors in quantitation arising due to the use of housekeeping proteins and associated normalization issues, a growing number of journals in the western blotting field have forced authors to withdraw published reports, issue errata, or have their publications rejected (Neill 2009). Journals are now requiring strict adherence to the use of internal controls and are mandating the use of imaging techniques that yield linear signal ranges and report linear dynamic range of signal (see guidelines for reporting life sciences research from Nature Publication Group 2013 and guidelines from Journal of Biological Chemistry). Stain-free technology fits perfectly in this new world of western blotting as it satisfies all the requirements for carrying out these new protocols and is considered an effective strategy to improve accuracy of western blots (Ghosh et al. 2014).
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