A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography

Here we describe a proteomic approach to detect fibrinolytic enzymes from the culture supernatant of Bacillus subtilis 168. Following isoelectric focusing without dithiothreitol, two gels, one for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the other for zymography, were run in parallel. After silver staining of SDS-PAGE and activity staining of zymography gel, the two gels were superimposed to detect protein spots that coincided with clear zones on the zymography gel. We identified four protein spots and characterized them with matrix-assisted laser desorption/ionization mass spectrometry. Database search revealed that four spots contained at least one of the extracellular serine proteases such as WprA and Vpr. This combined method of two-dimensional gel and zymography can be used as a powerful tool to detect proteases from various organisms.     

Silver staining of high molecular weight proteins on large-pore polyacrylamide gels

A large-pore gel for electrophoresis in the presence of sodium dodecyl sulfate, composed of 2.55% polyacrylamide crosslinked with 2.75% methylenebisacrylamide, is described. This gel has a resolving power for very high molecular weight proteins and can be stained with silver. The gel is suitable for fractionation of factor VIII/von Willebrand factor directly from plasma samples. Visualization by silver staining revealed a series of covalently bound multimers with molecular weights of up to 8 X 10(6). The procedure described should be useful also for studies on other very high molecular weight proteins and nucleic acids.     

一种改进的双向电泳染色方法

本文涉及了双向电泳过程中的染色方法,即先用考马斯亮蓝染色,将胶上可见蛋白切下再银染的方法。这种方法可最大限度的减少胶中蛋白质点的损失,不仅避免了单一用考马斯亮蓝染色由于灵敏度不高而导致的低丰度蛋白的损失,也避免了单一用银染而使高丰度的蛋白因染色过度导致的损失。同时两种传统的染色方法结合完美,形成的新方法经济实用。     

Highly Selective Acridine and Ethidium Staining of Bacterial DNA and RNA

The acridine dyes acridine orange (AO) and coriphosphine O (CPO) and ethidium bromide (EtBr) were used to stain bacterial digests after electrophoresis in native and denaturing (SDS) polyacrylamide gels and were shown to stain DNA and RNA preferentially over other subcellular components in the gels. Vegetative cell digests of Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus showed intense staining of DNA with AO and CPO near the top of the gel, but little or no staining of other cellular constituents. EtBr stained both DNA and RNA in the gels. Protein standards and non-nucleic acid cellular constituents stained faintly with high concentrations (> 100 μM) of AO, lower concentrations (13.9 μM) of CPO, and did not stain with 0.5 μg/ml EtBr in denaturing gels. The complete set of cellular biochemicals was visualized by silver staining, while the protein subset was detected by Coomassie blue staining. The highest concentrations of AO (120 μM) and CPO (13.9 μM) were shown to detect purified DNA in gels with a sensitivity in the range of 25–50 ng per band. This work demonstrates the specificity of acridine and ethidium dyes for nucleic acids, while illustrating the level of non-nucleic acid-specific interactions with other cellular components by staining of electrophoretically separated cellular components in a gel matrix.     

简单快速的DNA银染和胶保存方法

本文介绍了一套简单快速的DNA银染以及胶保存的方法,整个过程仅需10~15分钟,而且背景浅,条带清楚,灵敏度高,稳定性好。胶保存采用双层玻璃纸夹心法,可长久地保存胶显色时的原貌。以常规PAG胶检测和HLA的SSCP分型为例,利用该套方法进行了银染以及胶的保存,均得到了满意的结果。该方法具有推广价值。 Abstract:This paper introduced the simple and rapid methods of silver staining and gel preservation.It was taken only about 10 and 15 minutes to stain a gel.The background of gel was light,the bands were clear,the sensibility was high and the stabilization was well by the method of silver staining.The gel preservation adopted a method named two-layer transparent plastic paper "Sandwich" which could keep the gel with primitive colors for a long time.The methods were used on PAG checking and SSCP typing of HLA and the results were satisfactory.The set of methods are expected to be widely used in laboratories.     

Detection of dextransucrase and levansucrase on polyacrylamide gels by the periodic acid-Schiff stain: staining artifacts and their prevention

One use of the periodic acid-Schiff (PAS) stain is to detect dextransucrase and levansucrase activities on polyacrylamide gels by staining their polysaccharide products, dextran and levan. When gels with heavy dextran or levan bands were PAS stained, proteins other than dextransucrase and levansucrase also were stained, and a high background developed during storage. The staining of proteins other than dextransucrase and levansucrase is caused by the diffusion of the periodate-oxidized carbohydrate before and after staining. This diffusion could be greatly slowed, and the staining artifact decreased, by following the PAS stain by a crosslinking treatment of the carbohydrate-dye complex. Protein staining artifacts could be prevented by using chymotrypsin to remove the protein from the gel at the stage after polysaccharide synthesis but before the PAS stain.     

In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis.

We examined the different steps necessary for the enzymatic digestion of proteins in the polyacrylamide matrix after gel electrophoresis. As a result, we developed an improved method for obtaining peptides for internal sequence analysis from 1-2 micrograms of in-gel-digested proteins. The long washing-lyophilization-equilibration steps necessary to eliminate the dye, sodium dodecyl sulfate, and other gel-associated contaminants that perturb protein digestion in Coomassie blue-stained gels have been replaced by washing for 40 min with 50% acetonitrile, drying for 10 min at room temperature, and then rehydrating with a protease solution. The washing and drying steps result in a substantial reduction of the gel slice volume that, when next swollen in the protease solution, readily absorbs the enzyme, facilitating digestion. The Coomassie blue staining procedure has also been modified by reducing acetic acid and methanol concentrations in the staining solution and by eliminating acetic acid in the destaining solution. The peptides resulting from the in-gel digestion are easily recovered by passive elution, in excellent yields for structural characterization. This simple and rapid method has been successfully applied for the internal sequence analysis of membrane proteins from the rat mitochondria resolved in preparative two-dimensional gel electrophoresis.     

A comprehensive evaluation of imidazole‐zinc reverse stain for current proteomic researches

In this paper, we comprehensively evaluated the capability of imidazole‐zinc reverse stain (ZN) in comparative proteomics. Three commonly used protein gel staining methods, including silver (SN), SYPRO Ruby (SR), and CB stain were investigated alongside for comparison purpose. A transparency scanning procedure, which may deliver more even and contrasting gel images, was found best for documenting ZN stained gels. Our results showed that ZN was more sensitive than SN, SR, and CB. It may reveal as few as 1.8 ng of proteins in a gel. Moreover, ZN was found to provide a linear dynamic range of staining for revealing proteins up to 140 ng, and show an insignificant staining preference. To analyze a ZN stained 2‐D gel image that generally comprises an apparent but even background, the Melanie 4 software was found more suitable than others. Furthermore, ZN demonstrated an equivalent or better MS compatibility than the other three staining methods. Intense and comprehensive MS profiles were frequently observed for ZN stained gel spots. Approximate two‐third of ZN stained gel spots were successfully identified for protein identities. Taken together, our results suggest that the prompt, cost effective and versatile ZN is well suited for current proteomic researches.     

Preparation of human heart for laser microdissection and proteomics

Proteomics generates information on expressed proteins, and laser microdissection (LMD) is a method that allows enrichment of specific cell types from complex heterogeneous tissue. Together they provide a powerful tool for functional genomic research. Here, we have investigated (i) the effects of fixation and staining on cardiac proteins separated by two-dimensional gel electrophoresis (2-DE) and (ii) feasibility of using LMD to separately prepare myocytes and blood vessels for 2-DE gel analysis. This is the first such study of human heart. The effect of fixation (ethanol or acetone), staining with haematoxylin and eosin in the presence and absence of xylene, and antibody staining was investigated. Proteins were separated by 2-DE and spots detected by silver staining. Quantitative spot analysis showed that contractile proteins were preserved under all conditions, and no significant differences were found when the groups studied were compared with the control group. However, there were differences in the visual quality of the gel patterns. LMD provided enough protein from blood vessels and myocytes to run one large-format (18 x 24 cm) 2-D gel for each subset of cells. Collection of this material took 70 h (approximately 2800 blood vessels and 17,000 myocytes) and resulted in tissue-specific gel patterns for these two structures. In conclusion, the use of haematoxylin and eosin staining without xylene provided the best morphology and did not significantly affect protein spot number.     

Silver stain for detecting 10-femtogram quantities of protein after polyacrylamide gel electrophoresis

A rapid and highly sensitive silver stain and color stain were developed for visualizing proteins. The procedure is simple and the bands were clear. This silver stain detects 100 pg quantities of proteins. In order to stain quickly, sensitively, and sharply a protein matrix in a gel, the repeated shrinkage and swelling gel was developed with a hyper- and hypotonic solution to remove the sodium dodecyl sulfate (SDS) from SDS-protein complex and to generate influx of staining solution into the gel. We have found that the silver staining method with the repeated exposure to hyper- and hypotonic solution and a narrow well produced 10 fg order of proteins.     

Dodecyl maltoside-sodium dodecyl sulfate two-dimensional polyacrylamide gel electrophoresis of chloroplast thylakoid membrane proteins

A two-dimensional electrophoretic system has been developed for the separation of chloroplast thylakoid membrane proteins. This system incorporates nondenaturing polyacrylamide gel electrophoresis in the presence of the nonionic detergent dodecyl-beta-D-maltoside in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Thylakoid membranes isolated from Spinacia oleracea were solubilized in 1.0% dodecyl-beta-D-maltoside and separated in 4-7% linear acrylamide gradient tube gels which contained 0.05% dodecyl-beta-D-maltoside. After electrophoresis, the tube gels were equilibrated with a sodium dodecyl sulfate-containing equilibration buffer and applied to a 12.5-20% acrylamide linear gradient gel. The Lammelli buffer system was used in both dimensions. The two-dimensional gels were analyzed by staining sequentially with 3,3',5,5'-tetramethylbenzidine-H2O2, Coomassie blue, and silver staining. A number of protein components were identified on "Western blots" of these two-dimensional gels by immunological localization. Membrane protein complexes such as the light-harvesting chlorophyll a/b protein complex, photosystem I, photosystem II, the cytochrome b6/f complex and ribulose bisphosphate carboxylase appear to migrate as essentially intact complexes in the first dimension and appear as vertical series of resolved subunits in the second dimension. This technique complements isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis in providing additional information concerning the subunit composition of membrane protein complexes and may prove to be of general utility for studying the protein composition of other membrane systems.     

一种鉴定过氧化氢酶活性的铁染色法

采用聚丙烯酰胺凝胶电泳(PAGE)技术,建立了一种新的鉴定过氧化氢酶活性的方法──铁染色法．结果表明,细菌过氧化氢酶及牛肝过氧化氢酶显示单一的酶活性带．该方法操作简单、快速、灵敏度高、专一性强.是一种切实可行的鉴定过氧化氢酶活性染色法.     

Activity staining of glutathione peroxidase after two-dimensional gel electrophoresis

We have developed a method for rapid activity staining of proteins with glutathione peroxidase (GPx) activity after 2-D gel electrophoresis. After separating proteins extracted from yeast, or mouse red blood cells, by two-dimensional gel electrophoresis, SDS was removed and the gel was submerged in a Tris-HCl buffer containing glutathione and hydrogen peroxide, followed by incubation with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and phenazine methosulfate (PMS). After this proteins with GPx activity appeared as clear zones on a purple background. This relatively simple activity staining method could be useful for rapid screening of proteins with GPx activity in cell extracts.     

An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies

Unfortunately, conventional large-format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large-format two-dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide-based matrix has been developed that, upon polymerization, facilitates the handling of one-dimensional and two-dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer-reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high-molecular-weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro-Q Diamond phosphoprotein stain, Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, respectively.     

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.Download video file.^{(88M, mp4)}     

核酸染料Genefinder使用方法的比较

目的:使用核酸染料Genefinder检测琼脂糖凝胶中的核酸,通过比较预染样品法、胶内染色法和后染法三种染色方法的染色效果,了解该染料的染色特性,以期找到性能稳定,染色效果好的染色方法.方法:在琼脂糖凝胶电泳中,以不同的染色方法使用核酸染料Genefinder进行染色,对染色结果进行比较分析.结果:使用电泳后染色方法染色效果较好,胶内染色法次之,预染样品法效果最差.结论:核酸染料Genefinder会干扰DNA的迁移效率,因此,使用Genefinder进行电泳后染色是一种较好的染色方法.     

Detection of polyhydroxyalkanoate synthase activity on a polyacrylamide gel

This study presents a method to detect active polyhydroxyalkanoate (PHA) synthase on a polyacrylamide gel that combines the polyhydroxybutyrate (PHB) polymerization reaction with Sudan Black B staining. After separation of the protein samples on a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the slab gel was submerged in a buffer containing β-hydroxybutyryl-coenzyme A (3-HBCoA) as substrate and incubated at room temperature for in vitro PHB polymerization. The active PHA synthase catalyzed 3-HBCoA into the PHB polymer and was stained with Sudan Black B. The active PHA synthase appeared as a dark blue band. The activity staining was of high sensitivity, capable of detecting 3.9 ng (0.273 mU) of Cupriavidus necator H16 PHA synthase purified from recombinant Escherichia coli. The detection sensitivity of activity staining was comparable to that of Western blotting analysis. Furthermore, the high sensitivity of activity staining enabled specific detection of the active PHA synthase in the crude extract of wild-type strain C. necator H16. This study provides a rapid, sensitive, and highly specific method for detecting active PHA synthase in gel. The method could be applied to detecting PHA synthase from wild-type bacteria and to the process of enzyme purification.     

An improved formulation of SYPRO Ruby protein gel stain: comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation

SYPRO Ruby protein gel stain is compatible with a variety of imaging platforms since it absorbs maximally in the ultraviolet (280 nm) and visible (470 nm) regions of the spectrum. Dye localization is achieved by noncovalent, electrostatic and hydrophobic binding to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream proteomics techniques such as matrix-assisted laser desorption/ionisation-time of flight mass spectrometry is assured. The principal limitation of the original formulation of SYPRO Ruby protein gel stain, is that it was only compatible with a limited number of gel fixation procedures. Too aggressive a fixation protocol led to diminished signal intensity and poor detection sensitivity. This is particularly apparent when post-staining gels subjected to labeling with other fluorophores such as Schiff's base staining of glycoproteins with fluorescent hydrazides. Consequently, we have developed an improved formulation of SYPRO Ruby protein gel stain that is fully compatible with commonly implemented protein fixation procedures and is suitable for post-staining gels after detection of glycoproteins using the green fluorescent Pro-Q Emerald 300 glycoprotein stain or detection of beta-glucuronidase using the green fluorescent ELF 97 beta-D-glucuronide. The new stain formulation is brighter, making it easier to manually excise spots for peptide mass profiling. An additional benefit of the improved formulation is that it permits staining of proteins in isoelectric focusing gels, without the requirement for caustic acids.     

Regeneration of enzymatic activity after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and zinc acetate staining: the example of inositol 1,4,5-trisphosphate 5-phosphatase.

Regeneration of enzyme activity after sodium dodecyl sulfate-gel electrophoresis was investigated with a purified inositol 1,4,5-trisphosphate 5-phosphatase. In order to avoid silver or Coomassie blue staining, we have used zinc acetate. This staining procedure was sensitive, rapid, and reversible provided that zinc cations are chelated and activity is extracted after diffusion out of the gel. The method allows some gel lane staining and identification of the enzyme based on catalytic activity.     

A faithful double stain of proteins in the polyacrylamide gels with Coomassie blue and silver

The conditions for prior fixing of proteins in a gel in order to attain a greater degree of faithful silver staining and sensitivity were examined. Fixing with formaldehyde enhanced the retention of proteins in a gel, particularly basic proteins such as histones and ribosomal proteins. The gel, one stained with Coomassie blue and following the removal of the free dyes, is capable of undergoing silver staining, and, moreover, the prestain considerably enhanced the staining intensity of various proteins differing in basicity in subsequent silver staining. Coupling the formaldehyde fixation with Coomassie brilliant blue prestain afforded a reproducible and pronounced stainability of various proteins.