Advantages and disadvantages of silver staining of proteins in polyacrylamide gel

Sensitivity of protein staining with Serva blue G-250 (Coomassie brilliant blue G-250 analogue) in polyacrylamide gel was determined. It has been shown that protein staining with 0.1% Serva blue G-250 results in the recovery of 80 to 35 ng of single protein, that is almost 10 times higher than reported previously for Coomassie brill. blue G-250 (or R-250) staining. The comparison of the sensitivity of Serva blue G-250 protein staining in PAAG and AgNO3 has shown that AgNO3 staining was approximately 18-30 (but not 100 times, as it had been thought before) times more effective for the majority of proteins under study. Silver staining of some proteins, for instance ribonuclease and a number of retrovirus-specific structural proteins, was of lower efficacy. Thus, to obtain reliable results protein electrophoresis in PAAG should be followed by both staining procedures.     

失血性休克大鼠肝脏蛋白质的差异分析

 应用蛋白质双向凝胶电泳 (two-dimensional polyacrylamide gel electrophoresis, 2-DE) 技术,分析在急性重度失血性休克 (refractory hemorrhagic shock, RHS) 条件下,大鼠肝脏蛋白质组表达的差异.16 只雄性 Wistar 大鼠随机分成正常对照组 (sham hemorrhage shock, SHS) 和 RHS 模型组,每组 8 只.采用股动脉放血的方法制备模型,在规定时间内处死大鼠并分离肝脏,提取肝脏总蛋白质后进行 2-DE.运用 Image Master 2D Platinum v 5.0 凝胶图像分析软件对 2-DE 凝胶图像进行差异表达分析.有意义的差异蛋白质点用基质辅助激光解析电离飞行时间质谱进行肽质量指纹图谱分析,借助 Swiss-prot 数据库进行蛋白质搜索和鉴定.SHS 组和 RHS 组肝脏的 2-DE 图谱,分别平均识别到 698±11 和 700±13 个蛋白质点,SHS 组和 RHS 组肝脏间平均匹配率达88%～92 %.共发现 10 个差异有意义的蛋白质点,鉴定出了肿瘤抑制性抗原gp96、葡萄糖调节蛋白58、过氧还蛋白Ⅰ、细胞色素b5、谷胱甘肽转移酶、ATP合酶β亚单位、二磷酸果糖酶 B、三磷酸甘油醛脱氢酶等8种蛋白质.结果表明,以 2-DE 技术得到重复性和分辨率都较好的 2-DE图谱,并初步鉴定急性重度失血性休克后大鼠肝脏的差异表达蛋白质,为深入研究失血性休克的生理病理机制及寻找失血性休克预防和治疗的生物标志物提供了依据.     

Analysis of proteins in normal and regenerating rat liver using two-dimensional electrophoresis

The major proteins of homogenate, cytosol, nuclei and nuclear membrane extract from normal and regenerating rat liver were studied by two-dimensional electrophoresis with a view of detecting proteins involved in DNA replication regulation. Essential quantitative differences in three out of 200 polypeptides separated as spots and dyed with Coomassie R-250 on two-dimensional maps were revealed. The content of the p38 nuclear protein (Mr congruent to 38 kD, pI congruent to 4) increases 6-8-fold in the S-phase. The level of another nuclear protein, p50 (Mr congruent to 50 kD, pI congruent to 6.5) decreases 2-3-fold. The cytoplasmic protein p35 (Mr congruent to 35 kD, pI congruent to 8) also decreases 2-3-fold. Moreover, the p40 protein (Mr congruent to 40 kD, pI congruent to 6) whose content in the nuclei sharply rises up to 20 times after sham operation was revealed.     

Why does Coomassie Brilliant Blue R interact differently with different proteins? A partial answer

Dimethyl sulfoxide was found to be effective for extraction of Coomassie Brilliant Blue R-250 (Coomassie R) from stained proteins on polyacrylamide gel slices. A good correlation was found between the ability of different proteins to bind Coomassie R and their capacity for interaction with Coomassie Brilliant Blue G-250 (Coomassie G) in solution. Scatchard analysis showed that the number of Coomassie R ligands bound to each protein molecule is approximately proportional to the number of positive charges on the protein, about 1.5-3 dye molecules/charge.     

中国武汉地区葎草花粉变应原蛋白质组分的双向电泳分析

应用蛋白质双向电泳的分析技术对武汉地区生长的主要过敏原———葎草(Humulus scandensLour.)花粉的蛋白质组分进行了分析。应用三氯乙酸法提取葎草花粉总蛋白质,通过等电聚焦和第二向SDS-聚丙烯酰胺凝胶电泳分析获得了完整的葎草花粉全蛋白质图谱。应用专业分析软件(Im ageM aster 2D)对电泳图谱分析表明:通过等电聚焦和第二向SDS-PAGE电泳,有122个不同的蛋白质组分被检测出来,并进一步确定每个蛋白质相应的分子量、等电点和相对含量。研究中获得的高分辨率的双向电泳图谱是我国葎草花粉变应原蛋白质第一张完整的蛋白质图谱,将为今后葎草花粉中致敏蛋白质的检测、分离、基因克隆和变应原的标准化奠定基础。     

Analysis of total proteins in pollen of Humulus scandens Lour in Wuhan Region of China by two-dimensional electrophoresis

Total proteins in the pollen of Humulus scandens Lour,one of the most popular aeroallergens in China,were analyzed by two-dimensional electrophoresis in the current study.The proteins were extracted by Trichloracetic acid (TCA) method,and then separated by isoelectric focusing as the first dimension and SDS-PAGE as the second dimension.The spots of proteins were visualized by staining with Coomassie Brilliant Blue.After analysis with software (ImageMaster 2D),122 different proteins were detected;isoelectric point (pI),Molecular weight (MW) and relativevolume of each protein in the pollen were also discovered.This is the first high-resolution,two-dimensional protein map of the pollen ofHumulus scandens Lour in China.Our finding has built a solid foundation for identification,characterization,gene cloning and standardization of allergenic proteins in the pollen ofHumulus scandens Lour for further studies.     

Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set

We have expanded the reference set of proteins used in SELCON3 by including 11 additional proteins (selected from the reference sets of Yang and co-workers and Keiderling and co-workers). Depending on the wavelength range and whether or not denatured proteins are included in the reference set, five reference sets were constructed with the number of reference proteins varying from 29 to 48. The performance of three popular methods for estimating protein secondary structure fractions from CD spectra (implemented in software packages CONTIN, SELCON3, and CDSSTR) and a variant of CONTIN, CONTIN/LL, that incorporates the variable selection method in the locally linearized model in CONTIN, were examined using the five reference sets described here, and a 22-protein reference set. Secondary structure assignments from DSSP were used in the analysis. The performances of all three methods were comparable, in spite of the differences in the algorithms used in the three software packages. While CDSSTR performed the best with a smaller reference set and larger wavelength range, and CONTIN/LL performed the best with a larger reference set and smaller wavelength range, the performances for individual secondary structures were mixed. Analyzing protein CD spectra using all three methods should improve the reliability of predicted secondary structural fractions. The three programs are provided in CDPro software package and have been modified for easier use with the different reference sets described in this paper. CDPro software is available at the website: http://lamar.colostate.edu/ approximately sreeram/CDPro.     

Identification of specific proteins synthesized by type II pneumocytes in primary culture

Proteins from primary cultures of type II granular pneumocytes have been examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to identify type II cell-specific proteins. The distribution of Coomassie Blue-stained bands in preparations of cellular proteins, culture medium, lavage and lamellar bodies have been compared. The most prominent stained band in the serum-free medium from type II cell cultures (HS1; Mr 39900) corresponds to a major protein in acellular sedimentable (20000 g for 30 min) crude surfactant obtained from rat lungs by saline (0.9% NaCl) lavage. A second protein (HS2; Mr 12000) is also found both in type II cell-conditioned medium and in lavage. Neither rat serum nor donor calf serum (used in the isolation of the type II cells) contains a protein co-migrating with HS1 or HS2 proteins. HS1 is also found in Coomassie Blue-stained gels of cellular proteins and of lamellar bodies isolated from whole lungs. Cultures of type II cells incorporate [14C]phenylalanine into HS1 and HS2 as shown by autoradiography of sodium dodecyl sulphate/polyacrylamide gels of culture medium. Rat lungs perfused in situ incorporate [35S]methionine into HS1 in the lamellar body fraction. A third protein (HS3; Mr 47000) is observed only in autoradiographs of cell culture medium; no corresponding Coomassie Blue-stained band can be identified in medium, in cells or in lung lavage. No protein bands corresponding to HS1, HS2 or HS3 are found in conditioned media from pulmonary alveolar macrophages, rat fibroblasts or bovine aorta endothelial cells. Two-dimensional gel electrophoresis of HS1 shows a single polypeptide with an isoelectric point of 6.3; HS3 appears as a chain of spots with a range of isoelectric points from 6.3 to 6.6. HS2 has not been identified on two-dimensional gels. The amino acid composition of HS1 does not differ significantly from that of surfactant apoproteins studied previously; however, HS1 is not detected by glycoprotein stains, nor does it appear to be a subunit of a thiol-linked multimer.     

Alterations in surface proteins during myogenesis of a rat myoblast cell line.

Lactoperoxidase-catalyzed iodination and SDS-polyacrylamide gradient gel electrophoresis were used to examine the surface proteins of cultures of an embryonic rat myoblast cell line during myogenesis. We observed several consistent alterations in the proteins iodinated during the periods of alignment and fusion. In addition, we examined the surface proteins of cultures where fusion was inhibited by phospholipase C (PLC), and of cultures of several nonfusing variants of our original line. Many of the proteins which appeared during “normal” myogenesis were not seen in PLC-treated cultures, while the appearance and loss of three low molecular weight proteins were accelerated. The nonfusing variants often accumulated large amounts of many of the proteins which appeared during alignment in normal cultures. This accumulation was demonstrated by Coomassie blue stain intensities as well as by the extent of surface iodination. The three low molecular weight proteins were heavily iodinated in one class of variant, but did not disappear as in normal cultures. One protein of apparent molecular weight 66,000 (66K) was iodinated during alignment but was inaccessible during fusion. Coomassie blue staining of the gels showed that the actual appearance and disappearance of the 66K protein band from the membrane were coincident with alignment and fusion. While this band was not seen in fluorograms from gels of PLC-treated cultures and some of the nonfusing variants, a 66K band was invariably stained by Coomassie blue, and in PLC-treated cultures appeared to accumulate with time. In the variants there appeared to be a correlation between the availability of the 66K protein for iodination and the appearance of the low molecular weight proteins.     

Characterization of chloroplast thylakoid polypeptides in the 32-kDa region: polypeptide extraction and protein phosphorylation affect binding of photosystem II-directed herbicides

In order to distinguish between two photosystem II proteins with apparent molecular weights of about 32 kDa, mild extraction procedures were used to remove several thylakoid membrane components. A 32-kDa protein that stained intensely with Coomassie brilliant blue could be extracted from the thylakoid membranes without removing the 32-kDa herbicide receptor protein, which stained poorly with Coomassie brilliant blue. The nonextracted protein was readily detectable after in vivo polypeptide labeling with [35S]methionine or after in vitro covalent tagging with [14C]azidoatrazine. The procedures used to extract the intensely stained, 32-kDa polypeptide resulted in changes in herbicide-binding characteristics, presumably due to conformational changes in the herbicide-binding environment. Alterations of membrane surface charge by protein phosphorylation also influenced herbicide binding.     

Structural proteome of human colostral fat globule membrane proteins

Milk fat globule membrane (MFGM) contains proteins derived from the apical membrane of secreting epithelial cells of the mammary gland. Between 2-4% of total human milk protein content is associated with the fat globule fraction, as MFGM proteins. While MFGM proteins have very low classical nutritional value, they play important roles in various cell processes and defence mechanisms for the newborn. To date, fewer than 30 human MFGM proteins have been identified and characterized, either by immunological methods or by Edman sequencing and mass spectrometry. This study aimed to update the structural proteome of human colostral MFGM proteins and to create an annotated two-dimensional electrophoresis (2-DE) MFGM protein database available on-line. More than one hundred 2-DE spots derived from human colostral MFGM proteins were investigated by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and proteins were identified by three different software packages available on the web (PeptIdent, MS-Fit and ProFound); uncertain identifications were solved by nanoelectrospray ionization-ion trap mass spectrometry using SEQUEST software.     

Studies and critique of Amido Black 10B, Coomassie Blue R, and Fast Green FCF as stains for proteins after polyacrylamide gel electrophoresis.

The properties of amido black 10B (C.I. 20470), Coomassie blue R (C.I. 42660), and fast green FCF (C.I. 42053) as protein stains, along with a few comments on Coomassie blue G (C.I. 42655), are presented and dye impurities and their effects on protein-dye binding within gels are discussed. All three dyes produced metachromatic effects with some proteins. Problems encountered with long-term stability and fixation of certain maize seed proteins are reported along with procedures for overcoming them. The low solubility of Coomassie blue R in trichloroacetic acid prevented maximum staining and destaining within a reasonable time, whereas other solvents allowed diffusion of some proteins during staining. Coomassie blue R binds to proteins in much higher amounts than do amido black and fast green, which accounts for its sensitivity in detection of protein bands in gels. Procedures for obtaining maximum contrast with photographs are also outlined.     

Identification of proteins from non-model organisms using mass spectrometry: application to a hibernating mammal

A major challenge in the life sciences is the extraction of detailed molecular information from plants and animals that are not among the handful of exhaustively studied "model organisms." As a consequence, certain species with novel phenotypes are often ignored due to the lack of searchable databases, tractable genetics, stock centers, and more recently, a sequenced genome. Characterization of phenotype at the molecular level commonly relies on the identification of differentially expressed proteins by combining database searching with tandem mass spectrometry (MS) of peptides derived from protein fragmentation. However, the identification of short peptides from nonmodel organisms can be hampered by the lack of sufficient amino acid sequence homology with proteins in existing databases; therefore, a database search strategy that encompasses both identity and homology can provide stronger evidence than a single search alone. The use of multiple algorithms for database searches may also increase the probability of correct protein identification since it is unlikely that each program would produce false negative or positive hits for the same peptides. In this study, four software packages, Mascot, Pro ID, Sequest, and Pro BLAST, were compared in their ability to identify proteins from the thirteen-lined ground squirrel (Spermophilus tridecemlineatus), a hibernating mammal that lacks a completely sequenced genome. Our results show similarities as well as the degree of variability among different software packages when the identical protein database is searched. In the process of this study, we identified the up-regulation of succinyl CoA-transferase (SCOT) in the heart of hibernators. SCOT is the rate-limiting enzyme in the catabolism of ketone bodies, an important alternative fuel source during hibernation.     

Characterization of the cilia and ciliary membrane proteins of wild- type Paramecium tetraurelia and a pawn mutant

Cilia and ciliary membranes were isolated from axenically grown, wild- type Paramecium tetraurelia strain 51s and from the extreme pawn mutant strain, d495, derived from this parental strain. Over 60 protein bands having molecular weights of 15 to greater than 300 kdaltons were detected by Coomassie Blue staining of whole cilia proteins separated by one-dimensional SDS polyacrylamide gel electrophoresis. About 30 of these protein bands were visible in Coomassie Blue-stained membrane separations. About 60 bands were detected by silver staining of one- dimensional gels of membrane proteins. Differences between Coomassie Blue-stained separations of wild-type and pawn mutant strain d495 membrane proteins were seen in the quantity of a band present at 43 kdaltons. Radioiodination of cell surface proteins labeled approximately 15 protein bands in both wild-type and mutant cilia. The major axonemal proteins were unlabeled. Six membrane glycoproteins were identified by staining one-dimensional separations with iodinated concanavalin A and lentil lectin, two lectins that specifically bind both glucose and mannose residues. Two major neutral sugar species present in an acid hydrolysate of the cilia preparation were tentatively identified as glucose and mannose by gas chromatography of the alditol acetate derivatives.     

Never born proteins as a test case for ab initio protein structures prediction

The number of natural proteins although large is significantly smaller than the theoretical number of proteins that can be obtained combining the 20 natural amino acids, the so-called “never born proteins” (NBPs). The study of the structure and properties of these proteins allows to investigate the sources of the natural proteins being of unique characteristics or special properties. However the structural study of NPBs can also been intended as an ideal test for evaluating the efficiency of software packages for the ab initio protein structure prediction. In this research, 10.000 three-dimensional structures of proteins of completely random sequence generated according to ROSETTA and FOD model were compared. The results show the limits of these software packages, but at the same time indicate that in many cases there is a significant agreement between the prediction obtained.     

Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors

In Escherichia coli and other cell-based expression systems, there are critical difficulties in synthesizing membrane proteins, such as the low protein expression levels and the formation of insoluble aggregates. However, structure determinations by X-ray crystallography require the purification of milligram quantities of membrane proteins. In this study, we tried to solve these problems by using cell-free protein expression with an E. coli S30 extract, with G protein coupled receptors (GPCRs) as the target integral membrane proteins. In this system, the thioredoxin-fusion vector induced high protein expression levels as compared with the non-fusion and hexa-histidine-tagged proteins. Two detergents, Brij35 and digitonin, effectively solubilized the produced GPCRs, with little or no effect on the protein yields. The synthesized proteins were detected by Coomassie brilliant blue staining within 1h of reaction initiation, and were easily reconstituted within phospholipid vesicles. Surprisingly, the unpurified, reconstituted thioredoxin-fused receptor proteins had functional activity, in that a specific affinity binding value of an antagonist was obtained for the receptor. This cell-free translation system (about 1mg/ml of reaction volume for 6-8 h) has biophysical and biochemical advantages for the synthesis of integral membrane proteins.     

Biogenesis of Chromaffin Granules: Incorporation of Sulfate into Chromogranin B and into a Proteoglycan

The incorporation of [35S]sulfate into the soluble proteins of chromaffin granules was studied. Isolated bovine chromaffin cells were pulse-labeled with [35S]sulfate. The radioactively labeled products were characterized by one- and two-dimensional electrophoresis. Three proteins of chromaffin granules were preferentially labeled. One was identified by immunoprecipitation as chromogranin B (Mr 100,000). This result explains why during cellular synthesis the chromogranin B precursor is converted into a significantly more acidic protein. During chase periods, the newly synthesized chromogranin B was progressively degraded by endogenous proteases. A second labeled protein, much less labeled than chromogranin B, was identified as chromogranin A. The largest portion of the radioactive label was found in a heterogeneous component (Mr 86,000-100,000; pI 4.3-5.0). Digestion experiments with chondroitinase ABC demonstrated that this labeled component and a comigrating Coomassie Blue-stained spot were selectively degraded by this enzyme. This establishes that this component is a proteoglycan.     

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。     

The correlation between heat-shock protein accumulation and persistence and chilling tolerance in tomato fruit.

Heating tomato fruit (Lycoperiscon esculentum) for 48 h at 38 degrees C prevented chilling injury from developing after 21 d at 2 degrees C, whereas unheated fruit developed high levels of injury. Although the overall protein pattern as seen by Coomassie blue staining was similar from heated and unheated fruit, some high- and many low-molecular-mass proteins were observed in the heated fruit that were absent or present in reduced amounts in unheated fruit. When fruit wer injected with [35S]methionine at harvest and then heated, they accumulated high levels of specific radiolabeled proteins that could still be detected after 21 d at 2 degrees C. If the fruit were held at 20 degrees C after heating, the label in the proteins declined rapidly and these fruit were also sensitive to chilling injury. Hsp70 antibody reacted more strongly with proteins from heated and chilled fruit than with proteins from chilled fruit. Hsp18.1 antibody reacted strongly with proteins from heated fruit but not with those from unheated fruit. A 23-kD protein, highly labeled in heated fruit but not in unheated fruit, had its amino terminus sequenced. To our knowledge, this is the first report showing a relationship between the persistence of heat-shock proteins and chilling tolerance in a plant tissue.     

Schistosoma mansoni: protein composition and synthesis during early development; evidence for early synthesis of heat shock proteins

There are alterations in the proteins synthesized during different stages of development of Schistosoma mansoni. The protein profiles at different stages were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When stained by Coomassie blue, no significant differences were seen in protein profiles derived from cultured schistosomula from Days 0 to 6 and from adults. Newly synthesized proteins were detected by [35S]methionine incorporation. There were only a few differences in the protein profiles of schistosomula from Days 1 to 6 and from adults. Profiles derived from Day 0 schistosomula showed striking differences. Only a few proteins appear to be synthesized on Day 0 under these conditions. Schistosomula on Day 0 synthesized several minor proteins as well as a major protein of approximately 69,000 Da. This protein was immunoprecipitated by rabbit antiserum against bovine uncoating adenosine triphosphatase which recognizes the constitutive and induced 70,000 Da heat shock proteins in a wide variety of eukaryotic cells. More significant differences were observed when the newly synthesized proteins were analyzed by two dimensional gel electrophoresis. The profiles of newly synthesized proteins showed a specific repertoire of expression during the early stages of development in the parasite. A shift in temperature and medium during transformation from aquarium water to isotonic medium may initiate the synthesis of heat shock protein in these parasites.