RAMA stain: A fast,sensitive and less protein‐modifying CBB R250 stain

SDS‐PAGE and CBB staining are two of the most popular methods used for protein analysis. Although many reports that describe such staining methods have been published, these conventional protocols require several hours or days for staining and de‐staining. In this study we describe a recently developed, fast and sensitive CBB staining method that utilizes the staining solution of RAMA that consists of the low‐cost reagents: CBB R250, acetic acid, methanol and ammonium sulfate, and the destaining solution of water. Our method dose dependently detects 12 nanograms protein within 60 min and with a wide protein spectrum. Although the features of the dose‐dependent relationship depend upon protein amounts and protein types, for most of the protein samples tested, a linear relationship was observed in the region from 12 to 330 ng. Moreover, through further washing, the detection sensitivity of protein is enhanced and reaches a maximum at 1.4 ng and then gradually decreases in the de‐staining process. It has been shown recently through MS analyses that the sensitive colloidal CBB staining methods frequently result in artifactual methylations due to the strong acid and long contact during staining and the destaining processes. Such artifacts were reported to be reduced by the replacement of strong inorganic acid with acetic acid and because RAMA utilizes acetic acid and is in contact with the proteins for a short time during staining and de‐staining, it is expected that in vitro artifacts will be reduced. Finally, MS analyses of RAMA‐stained protein bands were revealed not to have been methylated.     

Western blot normalization: Time to choose a proper loading control seriously

Recent research has questioned the validity of housekeeping proteins in Western blot. Our present study proposed new ideas for Western blot normalization that improved the reproducibility of scientific research. We used the Gene Expression Omnibus (GEO) database and the web tool GEO2R to exclude unstable housekeeping genes quickly. In ischemic heart tissues, actin and tubulin changed significantly, whereas no statistically significant changes were observed in the expression of genes relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Besides, the reliability of GAPDH was further examined by Western blot. Additionally, unstable housekeeping genes were found in other animal models of cardiovascular medicine. We also found that sodium dodecyl sulfate and temperature significantly impacted the results of Ponceau S staining. Membranes stained with Ponceau S after immunodetection could avoid this interference, and the coefficients of variation for post-immunodetection staining are lower than those produced by GAPDH immunodetection. Overall, we described a new use of differential gene expression analysis and proposed a modified Ponceau S staining method, which provided researchers with a proper loading control for Western blot and hence could improve reproducibility in research.     

Classifying Human Haptoglobin Phenotypes on Native‐PAGE Using a Modified Coomassie Brilliant Blue R 250 Staining

There are three types of human Haptoglobin, Hp 1‐1, Hp 2‐1, and Hp 2‐2, each characterized by a distinct combination of the two α chains of the holoprotein. A modified Coomassie Brilliant Blue R 250 (mCBB‐R250) staining method for detecting the phenotypes of human blood haptoglobin molecules is presented. Addition of excess hemoglobin to the sample allowed specific formation of different haptoglobin‐hemoglobin complexes, which, in turn can be separated into distinctive migration pattern on native‐PAGE and stained by CBB‐R250. The typing results are consistent with that using Western blotting. In comparison to the existing methods for haptoglobin typing, our method is comparable in accuracy, and is easier to carry out. The results on typing of 29 plasma samples from Taipei Blood Center were also presented.     

β‐Actin as a loading control: Less than 2 μg of total protein should be loaded

For the purpose of data normalization in Western blot analysis, journal editors and reviewers usually require authors to reprobe the Western blot membrane with a β‐actin‐specific antibody after detecting the target protein. In most cases, however, β‐actin is overloaded, which results in a failure to detect differences in protein loading. In this study, we attempted to optimize the amount of protein loaded for β‐actin detection to permit suitable Western blot analysis data normalization. Our data suggest that less than 2 μg of total protein should be loaded when β‐actin is used as a loading control. We also suggest avoiding reprobing the membrane with a β‐actin‐specific antibody.     

Standard loading controls are not reliable for Western blot quantification across brain development or in pathological conditions

A frequently utilized method of data quantification in Western blot analysis is comparison of the protein of interest with a house keeping gene or control protein. Commonly used proteins include β‐actin, glyceraldehyde 3 phosphate dehydrogenase (GAPDH), and α‐tubulin. Various reliability issues have been raised when using this technique for data analysis—particularly when investigating protein expression changes during development and in disease states. In this study, we have demonstrated that β‐actin, GAPDH, and α‐tubulin are not appropriate controls in the study of development and hypoxic‐ischemic induced damage in the piglet brain. We have also shown that using an in‐house pooled standard, loaded on all blots is a reliable method for controlling interassay variability and data normalization in protein expression analysis.     

CBB staining protocol with higher sensitivity and mass spectrometric compatibility

Various CBB‐based methods for staining proteins separated by 2‐D gel electrophoresis were compared with regard to sensitivity and resolution. A modified Kang's CBB staining protocol, which we have modified, includes phosphoric acid in a concentration of 8% instead of the original 2%. This proved to be the best approach. Protein amounts as low as 2 ng and ∼2300 spots in the gel can be detected by employing this protocol. The modified procedure takes less time to carry out. Moreover, this practice is more sensitive and resolves more protein spots than most protocols reported to date and is compatible with subsequent mass spectrometric analysis.     

MALDI analysis of proteins after extraction from dissolvable ethylene glycol diacrylate cross‐linked polyacrylamide gels

Although the extraction of intact proteins from polyacrylamide gels followed by mass spectrometric molecular mass determination has been shown to be efficient, there is room for alternative approaches. Our study evaluates ethylene glycol diacrylate, a cleavable cross‐linking agent used for a new type of dissolvable gels. It attains an ester linkage that can be hydrolyzed in alkali conditions. The separation performance of the new gel system was tested by 1D and 2D SDS‐PAGE using the outer chloroplast envelope of Pisum sativum as well as a soluble protein fraction of human lymphocytes, respectively. Gel spot staining (CBB), dissolving, and extracting were conducted using a custom‐developed workflow. It includes protein extraction with an ammonia–SDS buffer followed by methanol treatment to remove acrylamide filaments. Necessary purification for MALDI‐TOF analysis was implemented using methanol–chloroform precipitation and perfusion HPLC. Both cleaning procedures were applied to several standard proteins of different molecular weight as well as ‘real’ biological samples (8–75 kDa). The protein amounts, which had to be loaded on the gel to detect a peak in MALDI‐TOF MS, were in the range of 0.1 to 5 μg, and the required amount increased with increasing mass.     

A new procedure for fast soft staining of BN‐PAGEs on photosynthetic complexes

We report a fast and sensitive procedure for blue native PAGE staining, in which the conventional staining step with CBB is avoided. After running, a short exposure to a mix of polar protic solvents (ethanol and acetic acid) leads to a fast and selective removal of the dye from the migration front and a specific binding to the protein bands, while the rest undergo a selective and complete background removal, leading to an intense contrast. This single‐step staining–destaining technique is useful in protein samples that bind colored cofactors such as photosystems, which can be selectively discerned by their characteristic green color. After the staining of such samples, the green color persists, while the other unpigmented protein complexes and the molecular standard remain CBB stained, creating a useful reference system for the assignment of the bands. The advantages and chemical basis of this staining procedure are discussed.     

Systematic comparison of technical details in CBB methods and development of a sensitive GAP stain for comparative proteomic analysis

Considering the importance of CBB staining in visualizing proteins in 2-DE gels, any improvement in the existing protocols with high sensitivity and good MS compatibility is of significant importance. In this study, we systematically evaluated the effects of different staining parameters on CBB methods by 1-DE and 2-DE, and demonstrated that G-250 was more suitable for visualizing low-abundant proteins as well as generating more spots than R-250, whereas R-250 had a superior capability for quick staining of high-abundant proteins. The staining produced by mixing G-250 and R-250 in different ratios showed similar sensitivity. Compared with acetic acid, phosphoric acid produced more protein spots. Ammonium-based stain demonstrated a superior sensitivity than the aluminum-based one. Based on these findings, a new protocol using CBB G-250, ammonium sulfate and phosphoric acid (GAP) was developed by incorporating the fixation, sensitization and staining procedures together. The comparison of GAP with other methods revealed that GAP generated more protein spots and had wider applications. The identification of 11 proteins demonstrated that GAP was not only compatible with MS but also obviously reduced in vitro protein modification, and thus could be a preferable protocol in the future proteomic analysis.     

Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples

Since most high throughput techniques used in biomarker discovery are very time and cost intensive, highly specific and quantitative analytical alternative application methods are needed for the routine analysis. Conventional Western blotting allows detection of specific proteins to the level of single isotypes while its quantitative accuracy is rather limited. We report a novel and improved quantitative Western blotting method. The use of fluorescently labelled secondary antibodies strongly extends the dynamic range of the quantitation and improves the correlation with the protein amount (r=0.997). By an additional fluorescent staining of all proteins immediately after their transfer to the blot membrane, it is possible to visualise simultaneously the antibody binding and the total protein profile. This allows for an accurate correction for protein load. Applying this normalisation it could be demonstrated that fluorescence-based Western blotting is able to reproduce a quantitative analysis of two specific proteins in blood platelet samples from 44 subjects with different diseases as initially conducted by 2D-DIGE. These results show that the proposed fluorescence-based Western blotting is an adequate application technique for biomarker quantitation and suggest possibilities of employment that go far beyond.     

Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex

SYPRO Ruby dye is a permanent stain comprised of ruthenium as part of an organic complex that interacts noncovalently with proteins. SYPRO Ruby Protein Gel Stain provides a sensitive, gentle, fluorescence-based method for detecting proteins in one-dimensional and two-dimensional sodium dodecyl sulfate-polyacrylamide gels. Proteins are fixed, stained from 3h to overnight and then rinsed in deionized water or dilute methanol/acetic acid solution for 30 min. The stain can be visualized using a wide range of excitation sources commonly used in image analysis systems including a 302 nm UV-B transilluminator, 473 nm second harmonic generation (SHG) laser, 488 nm argon-ion laser, 532 nm yttrium-aluminum-garnet (YAG) laser, xenon arc lamp, blue fluorescent light bulb or blue light-emitting diode (LED). The sensitivity of SYPRO Ruby Protein Gel Stain is superior to colloidal Coomassie Brilliant Blue (CBB) stain or monobromobimane labeling and comparable with the highest sensitivity silver or zinc-imidazole staining procedures available. The linear dynamic range of SYPRO Ruby Protein Gel stain extends over three orders of magnitude, which is vastly superior to silver, zinc-imidazole, monobromobimane and CBB stain. The fluorescent stain does not contain superfluous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. While peptide mass profiles are severely altered in protein samples prelabeled with monobromobimane, successful identification of proteins by peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry was easily performed after protein detection with SYPRO Ruby Protein Gel stain.     

Cell surface proteins of Candida albicans: preparation of extracts and improved detection of proteins

We have reexamined the detection of the components in a beta-mercaptoethanol and ammonium carbonate buffer extract of surface proteins of Candida albicans and the effects of postextraction manipulation of the extract on recovery of extract components. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), preferential staining of some moieties was observed when bands detected by a commercial silver staining method or a Coomassie Brilliant Blue (CBB) staining method were compared. Additional protein bands that were either not detected or poorly detected by a single method alone were readily observed by a combined silver-CBB staining method. This method also detected alterations in the profile of extracted proteins from organisms grown in the presence of galactose or hemoglobin rather than glucose. Two-dimensional electrophoresis (2-DE) gel analysis by double stain showed better detection of several acidic and basic protein spots. Less than 10% of the extract as determined by a dye-binding assay was lost following either or both lyophilization and dialysis. These manipulations of the extract did not change the protein profile following SDS-PAGE as determined by the combined staining or Western blot analysis of a 70 kDa protein. These observations suggest that soluble cell wall proteins are not unusually sensitive to procedures routinely used in protein purification. In addition, these studies suggest that a modified staining method that combines both silver stain and CBB stain provides improved detection of cell wall proteins compared to either method alone.     

Electrochemical Signal Tracing by Glucose Oxidase and Ferrocene Dually Functionalized Gold Nanoprobe for Ultrasensitive Immunoassay

A glucose oxidase (GOD) and ferrocene (Fc) dually functionalized gold nanoprobe was simply prepared for electrochemical immunoassay. By combination with sandwich immunoreaction at a carbon nanotube (CNT)‐based immunosensor and signal tracing of the nanoprobe through the Fc‐mediated GOD‐catalytic reaction, a new electrochemical immunoassay method was successfully developed. Both the multi‐enzyme signal amplification of the nanoprobe and the electron transfer promotion of the CNTs modified on the immunosensor greatly enhanced the signal response. Thus this method showed excellent analytical performance including ultrahigh sensitivity, wide linear range as well as good specificity, reproducibility, stability and reliability for human IgG measurement.     

Electrochemical Oxidation of Phenothiazine Derivatives at Glassy Carbon Electrodes and Their Differential Pulse and Square‐wave Voltammetric Determination in Pharmaceuticals

Abstract

Three 2,10‐disubstituted phenothiazines—chlorpromazine hydrochloride (CPM), thioridazine hydrochloride (TR) and propericiazine (PRC)—were electrochemically studied in various buffer systems at different pH values, using a glassy carbon electrode. The substances were electrochemically oxidized at potential range 0.55–0.75 V. The oxidation was reversible and exhibited diffusion‐controlled process. The mechanism of the oxidation process is discussed. According to the linear relation between peak current and concentration, differential pulse voltammetry (DPV) and square wave voltammetry (SWV) methods for quantitative determination of chlorpromazine and propericiazine in 0.1 M HClO₄, and thioridazine in pH 2 phosphate buffer, was applied. Both the repeatability and reproducibility of the methods were also determined for all studied substances. The developed procedures were successfully applied to the determination of chlorpromazine and thioridazine in pharmaceutical dosage forms.     

适于双向电泳分析的苹果叶片蛋白质提取方法

为了探索适用于双向电泳(2-DE)分析的苹果叶片蛋白质提取方法,比较了三氯乙酸(TCA)/丙酮沉淀法、二硫苏糖醇(DTT)/丙酮法、Tris-HCl提取法和改良的Tris-HCl提取法等4种蛋白质提取方法。以7 cm、pH 3～10的线性固相pH梯度(immobilized pH gradient,IPG)胶条作为第一向电泳,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)(12.5%的分离胶)作为第二向电泳,对提取物进行2-DE分离,采用银染显色。结果表明,上述4种方法在2-DE图谱上分别得到140,215,181和616个蛋白质点。其中以改良的Tris-HCl提取法得到的蛋白质点数最多,且背景清晰、图谱上没有明显的横纵条纹。为了进一步验证改良的Tris-HCl提取法的有效性,用18 cm、pH 3～10的线性IPG胶条和12.5%的分离胶对提取的苹果叶片蛋白质进行2-DE分离,考马斯亮蓝R-250染色,共检测到455个蛋白质点,其相对分子质量主要分布在14000～66000范围内,图谱背景清晰,再次证明应用该方法制备的样品适用于双向电泳分析,可用于苹果叶片的蛋白质组学分析。     

Design and synthesis of new fluorescent probe for rapid and highly sensitive detection of proteins via electrophoretic gel stain

A new fluorescent molecular probe, 2,2′‐(1E,1′E)‐2,2′‐(4‐(dicyanomethylene)‐4H‐pyrane‐2,6‐diyl)bis(ethene‐2,1‐diyl)bis(sodium benzenesulfonate) salt ( 1 ), possessing the cyanopyranyl moieties and two benzene sulfonic acid groups was designed and synthesized to detect proteins in solution and for high‐throughput SDS‐PAGE. Compound 1 exhibited no fluorescence in the absence of proteins; however, it exhibited strong fluorescence on the addition of bovine serum albumin as a result of intramolecular charge transfer. Compared with the conventional protocols for in‐gel protein staining, such as SYPRO Ruby and silver staining, 1 achieves higher sensitivity, even though it offers a simplified, higher throughput protocol. In fact, the total time required for protein staining was 60–90 min under optimum conditions much shorter than that required by the less‐sensitive silver staining or SYPRO Ruby staining protocols. Moreover, 1 was successfully applied to protein identification by mass spectrometry via in‐gel tryptic digestion, Western blotting, and native PAGE together with protein staining by 1 , which is a modified protocol of blue native PAGE (BN‐PAGE). Thus, 1 may facilitate high‐sensitivity protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.     

Fluorogenic Ag+–Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining

Silver staining, which exploits the special bioaffinity and the chromogenic reduction of silver ions, is an indispensable visualization method in biology. It is a most popular method for in‐gel protein detection. However, it is limited by run‐to‐run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) analysis; limitations that are largely attributed to the tricky chromogenic visualization. Herein, we reported a novel water‐soluble fluorogenic Ag⁺ probe, the sensing mechanism of which is based on an aggregation‐induced emission (AIE) process driven by tetrazolate‐Ag⁺ interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains.     

Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly‐horseradish peroxidase (PolyHRP)

Immunoassays such as ELISAs and Western blotting have been the common choice for protein validation studies for the past several decades. Technical advancements and modifications are continuously being developed to enhance the detection sensitivity of these procedures. Among them, Streptavidin‐containing poly‐horseradish peroxidase (PolyHRP) based detection strategies have been shown to improve signals in ELISA. The use of commercially available Streptavidin and antibodies conjugated with many HRPs (PolyHRPs) to potentially enhance the detection sensitivity in Western blotting has not been previously investigated in a comprehensive manner. The use of PolyHRP‐secondary antibody instead of HRP‐secondary antibody increased the Western blotting sensitivity up to 85% depending on the primary antibody used. The use of a biotinylated secondary antibody and commercially available Streptavidin‐conjugated with HRP or PolyHRP all resulted in increased sensitivity with respect to antigen detection. Utilizing a biotinylated secondary antibody and Streptavidin‐conjugated PolyHRP resulted in as much as a 110‐fold increase in Western blotting sensitivity over traditional Western blotting methods. Quantification of troponin I in rat heart lysates showed that the traditional Western blotting method only detected troponin I in ≥2 μg of lysate while Streptavidin‐conjugated PolyHRP20 detected troponin I in ≥50 ng of lysate. A modified blocking procedure is also described that eliminated the interference caused by the endogenous biotinylated proteins. These results suggest that Streptavidin‐conjugated PolyHRP and PolyHRP secondary antibodies are likely to be commonly utilized for Western blots in the future.     

Alternative methods for fixing and staining gliadins in polyacrylamide gels.

Staining efficiencies of Coomassie Brilliant Blue G-250 (CBB G-250) and Coomassie Brilliant Blue R-250 (CBB R-250) in various media were studied in efforts to reduce or eliminate requirements for trichloroacetic acid (TCA). Stained gels were compared with gels stained with CBB R-250 in 12% TCA and evaluated for overall stain and background. Because of qualitative effects, stain intensities of low- and high-mobility gliadins were also evaluated. Results indicated gliadins are fixed under a wide range of conditions, permitting adjustment of conditions to provide optimum staining. CBB G-250 and R-250 in tap water fixed and stained most gliadins. Best results were obtained with CBB G-250 in 2% TCA, in 2% TCA containing 5% sodium sulfate, and in 2% and 5% phosphoric acid containing 5% sodium chloride or 5% sodium sulfate. Gels stained in these media were more easily observed during staining and more easily destained than gels stained in CBB R-250 in 12% TCA.     

Studies on the Spectra of 4,5‐Dibromofluorescein with Proteins and its Analytical Application

4,5‐Dibromofluorescein(R) binding to proteins causes a decrease in the fluorescence maximum of R at 530 nm; the intensity of fluorescence quenching is directly proportional to protein content, Based on this, a new fast and simple fluorescence quenching method for the determination of proteins was developed. Under experimental conditions, the linear range of this assay is 0?15 mg/L and the detection limit is 40 μg/L. The method has been applied to the analysis of human serum samples and gave values close to those obtained by the Coomassie brilliant blue G‐250 (CBB G‐250) method, which indicated that the method is not only high in sensitivity but also reliable.