Protein extraction and comparison of stain protocols for analysis of two-dimensional electrophoresis gels

Protein extraction and the proteome of Lactobacillus delbrueckii subsp. bulgaricus were studied using different stains. The reversible silver staining technique was shown to be more sensitive than the irreversible silver stain. Coomassie colloidal was demonstrated to be as sensitive as reversible silver stain; however, the Coomassie colloidal blue solution developed a higher background and for sample preparation was more time-consuming.     

A sensitive staining method for detecting acidic polysaccharides in cellulose acetate and agarose gels

A sensitive staining method has been developed for the detection of acidic polysaccharides in cellulose acetate and agarose gels. The method is based on the precipitation of bovine serum albumin by acidic polysaccharides at acidic pH values and the subsequent staining of precipitated protein with amido black or Coomassie brilliant blue R-250 stains. The detection limit of acidic polysaccharides is 15-40 ng on cellulose acetate strips and 50-150 ng on agarose plates. The sensitivity of the described staining technique is of the same order for a wide range of acidic polysaccharides of different origin in contrast to Alcian blue and toluidine blue stains, which detect only mucopolysaccharides of animal origin at comparable levels. The method was also applied to the colorimetric quantitative determination of acidic polysaccharides after electrophoretic separation.     

Staining properties of bovine low molecular weight hydrophobic surfactant proteins after polyacrylamide gel electrophoresis.

Reports describing polyacrylamide gel electrophoresis patterns of bovine hydrophobic surfactant proteins are not consistent. In this study, we found unusual staining characteristics of these proteins that may explain some of these inconsistencies. Low molecular weight surfactant proteins extracted from bronchoalveolar lavage with organic solvent are partially delipidated with Sephadex LH-20 chromatography using chloroform and methanol. Fractions from the first protein peak are dried under nitrogen then subjected to SDS electrophoresis on 20% polyacrylamide gels. Under nonreducing conditions, silver staining identifies 5- and 26-kDa bands, and Coomassie blue identifies 6-, 12-, and 26-kDa bands. When gels are stained with Coomassie blue then silver, the 5- and 26-kDa bands stain with silver and 6- and 12-kDa bands remain stained with Coomassie blue. If gels are first stained with silver then Coomassie blue, similar results occur. We modified the silver staining protocol by treating gels with dithiothreitol or 2-mercaptoethanol after electrophoresis. With this modification, 5-, 6-, 12-, 26-, and also 17-kDa bands are identifiable. Using the modified protocol and restaining gels previously stained with silver, 6-, 12-, and 17-kDa bands that were not identified previously all became visible. In further experiments, protein bands of 6-, 12-, and 26-kDa that were identified by Coomassie blue were electroeluted under nonreducing conditions. After electrophoresis of the eluted 26-kDa protein, bands of 17-, and 26-kDa under nonreducing, and 8-kDa only under reducing conditions, were apparent by using the modified silver protocol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alcian blue as a protein stain for sodium alkyl sulfate-polyacrylamide gels: Application to analysis of polypeptide components of the human platelet

The cationic dye, Alcian blue, previously used as a glycoprotein-specific stain on cellulose acetate and polyacrylamide gels, was found to be capable of staining a variety of purified proteins and each of the components of the human platelet presently identifiable with Coomassie blue R or periodic acid-Schiff (PAS) reagent in sodium alkyl sulfate-polyacrylamide gel electrophoretic preparations. Evidence was obtained to indicate that staining of detergent-protein complexes by Alcian blue occurs by virtue of the affinity of the stain for accessible sulfate groups of detergent molecules, especially sodium tetradecyl sulfate, hydrophobically associated with polypeptide chains. Thus, Alcian blue fails to stain nonglycosylated proteins when pure sodium dodecyl sulfate (C₁₂) is used as the detergent, but does so readily when small quantities of sodium tetradecyl sulfate are also present. The advantages of using Alcian blue to determine platelet protein composition and to make quantitative comparisons between bands in sodium alkyl sulfate gels are discussed.     

Isolation of lumenal proteins from spinach thylakoid membranes by triton X-114 phase partitioning

The proteins present in the thylakoid lumen of higher plant chloroplasts have not been rigorously examined. In this communication we present a simple and rapid procedure for the isolation of the soluble proteins and extrinsic membrane proteins present in the thylakoid lumen from spinach. Our procedure involves extensive washing of the thylakoid membranes followed by Triton X-114 phase partitioning. When analyzed by one-dimensional polyacrylamide gel electrophoresis (PAGE), we obtain results which are very similar to those obtained by Kieselbach et al. using more classical methods [T. Kieselbach, A. Hagman, B. Andersson, W.P. Schroder, J. Biol. Chem. 273 (1998) 6710-6716]. About 25 major proteins are observed upon Coomassie blue staining. Upon two-dimensional isoelectric focusing-sodium dodecyl sulfate-PAGE and either Coomassie blue or silver staining, however, numerous other protein components are resolved. Our findings indicate that the total number of proteins (soluble and extrinsic membrane) present in the lumen may exceed 150.     

A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.

We have developed a highly sensitive stain for visualizing proteins in polyacrylamide gels. Our modification of the procedure for de Olmos' neural, cupric-silver stain is 100 times more sensitive than the conventional Coomassie blue stain (e.g., detection of 0.38 vs 38 ng/mm² of serum albumin), and is comparable to the sensitivity attained with an autoradiogram of ¹⁴C-methylated proteins following a 5-day exposure. This silver stain will be especially useful for analysis of patterns of proteins from tissue where attainment of the high specific activity of isotope labeling which is necessary to detect minor protein components is expensive, technically difficult or, as in humans, prohibited. In preliminary results with material such as unconcentrated cerebrospinal fluid, the silver stain revealed a complex pattern of proteins not visible with Coomassie blue.     

Quantitation of protein and DNA in silver-stained agarose gels

A silver stain for both proteins and DNA in agarose gels is described. Quantitation of proteins with this stain is possible, with individual proteins exhibiting characteristic responses, as observed with other stains. The advantage of the silver stain over Coomassie blue is its increased (50- to 100-fold) sensitivity, which allows samples containing very low protein concentrations to be analyzed without prior concentration. This silver stain, when applied to DNA, is at least as sensitive as ethidium bromide, and gives a linear response for the type of DNA and fragment sizes studied.     

Eosin Y staining of proteins in polyacrylamide gels.

A staining method is described in which various proteins in polyacrylamide gels can be stained by using eosin Y. After a brief incubation of a polyacrylamide gel in an acidic solution of 1% eosin Y, various proteins, including human erythrocyte membrane sialoglycoproteins which are not detectable by Coomassie blue R-250 (CB), can be detected with a sensitivity of 10 ng protein. This is far more sensitive than CB staining and is comparable to the sensitivity of silver staining. In a Western blot, the antigenicity of an eosin Y stained protein is retained. In addition, proteins on an immunoblot sheet can be detected by eosin Y staining. The method described is rapid, sensitive, and reproducible with various proteins in polyacrylamide gels and has the added advantage of also staining sialoglycoproteins.     

Fast and efficient method for detection and estimation of proteins

A quick, simple, inexpensive and sensitive method is described to stain and quantitate proteins on nitrocellulose papers. The proteins may be spotted or transferred from polyacrylamide gels by Western blotting. The procedure involves non-radioactive iodination of the polypeptides by chloramine T and potassium iodide followed by detection of bound iodine with starch. The method is more sensitive and much quicker than Coomassie brilliant blue staining and may be used for quantitation or detection of proteins in unknown samples. Another major advantage of this procedure is that ionic or nonionic detergents, although at higher concentrations causing the sample to disperse more broadly in the membranes, do not affect the staining procedure. Further, this method may be used for detection of proteins bound to papers that have high affinity for proteins such as the Zeta probe membranes.     

Two-dimensional gel electrophoretic resolution of the polypeptides of rat liver mitochondria and the outer membrane

The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.     

A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.     

微量蛋白检测中不同银染方法的比较与分析

随着生物化学技术的不断发展,作为检测SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)中微量蛋白的银染方法也在不断改进和发展.采用4种不同的银染方法检测不同含量的牛血清白蛋白,结果显示单纯的银染过程中如果使用戊二醛固定会使蛋白检出更快速灵敏,而结合考马斯亮蓝的复合银染则较单纯银染灵敏度提高了5～7个数量级.     

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 quantitative determination of the relative amount of histones in polyacrylamide gel by silver stain

On the basis of scanning densitometry of the stained gel, the conditions for the quantitative determination of individual histones by silver was examined and compared with the dye-staining method, in terms of higher sensitivity and faithful quantitation. Fixation with formaldehyde, coupled with simultaneous prestaining with Coomassie brilliant blue (CBB), was found to be most suitable. Prior fixation in acidic alcohol alone failed to stain the histones accurately, but this failure could be partly alleviated by prestaining with CBB. Although the sensitivity for detecting histones by silver staining is lower than that for neutral proteins by about 10-fold, it is at least 10-fold higher than the CBB stain.     

Analysis of differences between coomassie blue stain and silver stain procedures in polyacrylamide gels: conditions for the detection of calmodulin and troponin C

It is reported that the conditions used in some silver stain procedures can fail to detect calmodulin, troponin C, and other proteins with similar physical properties. Conditions are described that allow the reproducible detection of these proteins. Two phenomena are described: (1) lack of protein staining when treatment with glutaraldehyde is omitted from the protocol, and (2) loss of small proteins from the gel matrix during prolonged washing procedures. These data directly demonstrate that the use of some silver staining protocols can result in misleading data in biological studies and provide an explanation for at least one class of proteins of how silver staining and Coomassie blue staining of gels can give different results.     

A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels

We have simplified the highly sensitive silver stain of R. C. Switzer III, C. R. Merril, and S. Shifrin (1979, Anal. Biochem.98, 231–237) for visualizing proteins in polyacrylamide gels. We have reduced the number of steps in the procedure from 10 to 6, simplified the reagents in each step, and reduced the amount of silver required by a factor of 10, thus greatly reducing the expense of the procedure. In common with the original silver stain, our procedure is 100 times more sensitive than Coomassie brilliant blue and is comparable in sensitivity to radioautography of radioactively labeled proteins.     

A rapid sensitive silver stain for polypeptides in polyacrylamide gels

The use of silver to detect polypeptides was originally achieved by modifying tissue stains. By adapting methods of photochemistry we have developed a new silver stain for polypeptides which is nearly as sensitive but much more efficient than these earlier procedures. The new silver stain utilizes only three solutions and allows protein patterns to be visualized within 50 min. Its sensitivity is 100 times that of the Coomassie blue stain.     

A sensitive silver stain for proteins in agarose gels

A silver stain for proteins in agarose gels which is at least 10 times as sensitive as Coomassie blue is described. The method is simple to use and is particularly useful for the study of protein bands in the gamma region on electrophoresis of fluids such as cerebrospinal fluid in which the protein concentration is low. It readily detects bands of IgG containing 20 to 40 ng/band (approx 3 to 6 ng of IgG/mm2 of gel).     

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.     

家蚕丝腺蛋白质组学研究方法的建立

通过高精度的双向电泳技术对家蚕中部丝腺组织的蛋白质进行分离,采用基质辅助激光解析电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtimeofflightmassspectrometry,MALDI-TOF-MS)对其中一些表达量较高的蛋白点进行鉴定,并利用GPMAW(GeneralProtein/MassAnalysisforWindows)软件结合家蚕基因组预测的蛋白质数据库构建本地的肽质量指纹图谱数据库,对所得到的肽质量指纹图谱进行分析。研究发现,经过双向凝胶电泳及其图象分析技术,硝酸银染色和考马斯亮蓝染色分别能分离出500个以上和100个以上的蛋白点。这些蛋白质点主要集中在分子量15~90kD区域,等电点pH3·5~7之间。MALDI-TOF-MS鉴定的25个考染蛋白点中有60%以上的PMF(PeptideMassFingerprint)的信号峰较强。在数据库检索过程中,利用家蚕肽质量指纹数据库所得检索结果与在Mascot的检索结果相比,前者不仅能够准确鉴定出一些已有研究报道的蛋白,从而验证检索方法的可行性,而且还能够对一些已经被家蚕基因组数据库所预测但未曾报道的新蛋白质进行鉴定,从而建立了一整套适合于家蚕蛋白质组研究的方法,并为其它绢丝昆虫蛋白质组研究提供了重要参考。