两种织纹螺肌肉蛋白的电泳鉴别

利用聚丙烯酰胺凝胶电泳(SDS-PAGE)对有毒的光织纹螺(Nassariusrutilans)和无毒的西格织纹螺(Nassariussiquinjorensis)的肌肉蛋白做鉴别分析。实验采用四种不同抽提液对肌浆蛋白和肌原纤维蛋白进行提取。肌肉蛋白经电泳后以考马氏亮蓝染色并对蛋白图谱作对比分析。根据蛋白条带的数量、迁移率及浓度等特点,比较得出两种织纹螺的差异条带。综合比较得出,前者的肌肉蛋白特异条带为34.9kDa、32.7kDa,后者的特异条带为36.0kDa。结果表明,以SDS-PAGE电泳法可实现对该两种织纹螺的初步鉴别。     

Differentiation of Staphylococci from Iberian dry fermented sausages by protein fingerprinting

The Staphylococci populations in different types of Iberian dry fermented sausages from central-west Spain were identified. A simple electrophoretic method of whole-cell proteins and extracellular protein profiling was evaluated for speed of identification. This study was correlated with a 16S rRNA gene sequence analysis and biochemical identification by API Staph. A total of 81 isolates were identified by SDS-PAGE of the whole-cell proteins. These showed stable profiles in the range 99-14kDa that were clearly different for the different species, and were grouped into clusters together with the profiles of the eight reference strains. SDS-PAGE of the extracellular protein extracts provided additional characteristic banding patterns for the characterization of the Staphylococcus species present. The whole-cell SDS-PAGE showed that the predominant species was Staphylococcus saprophyticus (61.7%) followed by Staphylococcus aureus (19.7%). The identifications were confirmed by the 16S rRNA gene sequencing and by a BLAST search of the GenBank database. However, the API Staph biochemical identifications were frequently erroneous at the species level. In sum, SDS-PAGE analysis showed itself to be rapid and accurate in identifying the most commonly encountered Staphylococcus isolates in dry fermented sausages.     

Characterization of protein components of natural and heat-treated milk fat globule membranes

The proteins associated with the milk fat globule membrane (MFGM), isolated from early, mid and late season whole milks, were characterized using one- and two-dimensional SDS-PAGE under reducing and non-reducing conditions. In some experiments, MFGM separated from fresh whole milk was suspended in simulated milk ultrafiltrate (SMUF) and heated at various temperatures and times. SDS-PAGE under reducing conditions followed by staining with Coomassie blue showed the presence of about 37 protein bands, ranging in molecular weight from 15 to 200 kDa. SDS-PAGE under non-reducing conditions showed only about 25 distinct bands and the intensity of xanthine oxidase and butyrophilin bands was much less, while the intensity of PAS 6/7 band was similar compared with the reduced SDS-PAGE. Two-dimensional SDS-PAGE showed that the protein complexes that remained at the top of non-reducing gel were resolved into mostly xanthine oxidase and butyrophilin with a small proportion of PAS 6. These results indicate that xanthine oxidase and butyrophilin may be complexed via intermolecular disulfide bonds in the natural MFGM, although it is not possible to differentiate the individual protein distributions within these aggregates. It was found that the total protein content (mg/g fat) of MFGM and the percentage of xanthine oxidase and butyrophilin in early and late season MFGM were higher than that of mid season MFGM. In heated samples (above 60°C), xanthine oxidase and butyrophilin interacted further to form higher molecular weight protein complexes, while PAS 6/7 was relatively heat stable.     

不同添加物对大麦发芽过程中热稳定蛋白含量的影响

为了解发芽过程中热稳定蛋白动态变化趋势,以澳麦为原料,模拟工业制麦条件,系统分析了麦芽制备过程中热稳定蛋白分布变化。通过在浸麦最后一天添加Mg2+、K+、赤霉素(GA3)调节蛋白酶酶活及分解。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)电泳和考马斯亮蓝法(Bradford法),研究添加金属离子及赤霉素后热稳定蛋白分布及含量。结果表明,麦芽中热稳定蛋白分子质量主要分布在约40 ku和10 ku两个区域。经制麦后,40 ku分子质量蛋白质含量增加,10 ku附近蛋白质含量变化不明显。Mg2+和赤霉素添加降低了热稳定蛋白含量,K+有利于热稳定蛋白含量增加。     

Biochemical estimation of hepatitis B surface antigen in recombinant yeast

Purified recombinant hepatitis B surface antigen separated on polyacrylamide gels in the presence of sodium dodecyl sulphate has a very low staining index with Coomassie blue relative to a number of standard proteins. In contrast the protein stains better than average with silver nitrate. This property has been used to develop a semi-quantitative method of estimation of recombinant surface antigen in extracts of Saccharomyces cerevisiae producing this protein. The method can be used to follow purification protocols. It is quick, simple and since it measures the surface antigen biochemically, is independent of the aggregation state or conformation of the protein, a factor which can affect enzyme-linked immunoassays which rely on antigen-antibody interactions.     

Electrophoretic Identification of Raw and Cooked Shrimp using Various Protein Extraction Systems

Proteins were extracted from raw or cooked pink (Penaeus duorarum), white (Penaeus setiferus) or rock shrimp (Sicyonia brevirostris) with five different solutions: water, water homogenate adjusted to pH 8.0, 0.1M NaCl, 1% SDS, or 8M urea. Each extract from each species was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Water extraction showed highly species-specific banding patterns for raw shrimp, while patterns for SDS extracts were not as species-specific. However, SDS extracts provided the greatest information on species variation of cooked shrimp. SDS-PAGE was useful in distinguishing shrimp of different genus. This technique was tested and proven in a blind study to be useful for species identification and detection (within 10% of actual amount) of fabricated products.     

国内外不同品种啤酒大麦萌发过程中蛋白质的变化

采用9种国内外啤酒大麦为材料,以考马斯亮蓝G-250方法榆测其萌发过程中清蛋白、球蛋白、醇溶蛋白和谷蛋白含量的动态变化,并进行SDS-PAGE电泳分析.结果表明:大麦萌发过程中,清蛋白含量逐渐升高,平均增加2.116倍;球蛋白含量先升高后降低,萌发后比萌发前略高;醇溶蛋白和谷蛋白的含量逐渐降低,分别平均减少1.983和2.167倍.国内外不同品种啤酒大麦的清蛋白和球蛋白组成基本相同,醇溶蛋白存在显著的差异,谷蛋白也有一定的差别,因此醇溶蛋白可以作为鉴定大麦不同品种和品质的依据之一,谷蛋白也可以作为一种辅助的鉴别依据.     

Preliminary Studies on SDS-PAGE and Isoelectric Focusing Identification of Sturgeon Sources of Caviar

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) analyses of caviar were used to identify species of American, European and Asian sturgeon. Caviar samples of American and Gulf of Mexico sturgeon showed distinctive protein banding patterns on SDS-PAGE. IEF gels containing 20% pH 3-10 and 80% pH 4-6.5 ampholyte enabled differentiation of sturgeon species. IEF was more reliable than SDS-PAGE for identification of sturgeon species.     

Identification of Red Snapper (Lutjanus campechanus) using Electrophoretic Techniques

Isoelectric focusing (IEF), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional (2-D) gel electrophoresis were employed to produce protein profiles for species identification of red snapper and 11 other fish species. Comparing the distinctive patterns of water-soluble sarcoplasmic proteins for each species on IEF and SDS gels, red snapper could be identified. IEF gels of ampholyte mixture of 20% pH 3–10 and 80% pH 4–6.5 resolved better than gels with ampholyte ranging from pH 3–10, 4–6.5, 6–8, or 5–6 for species identification. The 10 and 12.5% SDS-PAGE gels produced more distinctive protein profiles for identification than 7.5 and 15% gels. Thus, these techniques could be applied to identify fish species.     

USE OF A MODIFIED UREA GEL ISOELECTRIC FOCUSING METHOD FOR SPECIES IDENTIFICATION OF RAW OR BOILED WHITE,PINK, AND ROCK SHRIMP1

Isoelectric focusing (IEF) polyacrylamide gel containing an 80% pH 4–6.5 and 20% pH 3–10 ampholyte mixture greatly improved protein banding pattern for species identification of water extracts of raw pink, white and rock shrimp compared with the system using only the pH 3–10 range ampholyte. Identification of a specific species in mixture samples was achieved by the detection of water-extractable shrimp specific protein bands present in the gel. Sodium dodecyl sulfate (SDS) was a better protein extractant than water for cooked shrimp. Both water and SDS extracts of cooked shrimp showed specific protein banding patterns and improved resolution for species identification.     

Characterisation of the S-Carboxymethyl Extract of Wool Using Small Format Alkaline Urea-PAGE/SDS-PAGE Followed by Silver Staining

Abstract

Alkaline urea-PAGE/SDS-PAGE, when used in a novel format which was significantly smaller than that employed by earlier workers, and when followed by silver staining, resulted in the detection of excellently resolved protein components from S-carboxymethyl reductive extracts of very small wool samples, even samples as small as an individual wool fibre. The silver stained two-dimensional gel patterns exhibited significant improvements compared to the fluorograph gel patterns of earlier workers based on the presence of radiolabel incorporated within the S-carboxymethyl moiety. The silver staining resulted in the visualisation of numerous protein components, and in the region of the gel pattern expected to contain the high-sulfur protein fraction, there appeared more major components than the number of high-sulfur protein components usually displayed in fluorograph patterns. The relative amount of the high-sulfur protein fraction in the final silver stained gel pattern could be boosted, if desired, if the gel was loaded not with the whole wool extract but with the filtrate resulting from the passage of the whole extract through a centrifugal ultrafilter.     

牛乳中αs、β-酪蛋白组分的分离

以新鲜脱脂牛乳为原料,在碱性条件下添加钙盐进行选择性沉淀分离αs-、β- 酪蛋白组分,并用十二烷基磺酸钠- 聚丙烯酰胺凝胶电泳(SDS-PAGE)法测定分离效果。从CaCl2 溶液终浓度、冷却温度和反复溶解﹑沉淀次数三个方面对αs-、β- 酪蛋白的分离效果进行研究。结果表明：CaCl2 溶液终浓度0.065mol/L,冷却温度2℃,经3 次反复溶解﹑沉淀,分离得到的αs- 酪蛋白组分纯度较高,可达83.33%,β- 酪蛋白组分纯度为109.53%。     

Species Identification of Raw and Boiled Shrimp by a Urea Gel Isoelectric Focusing Technique

Isoelectric focusing (IEF) using 9.2M urea and 6.2% (v/v) ampholytes in a polyacrylamide gel was used to separate protein banding patterns for species identification of pink, white and rock shrimp. A 17-hr focusing time was used. IEF of water extracts of raw shrimp showed excellent banding patterns useful for distinguishing each shrimp species. For cooked shrimp, water and sodium dodecyl sulfate (SDS) were better protein extractants. Detection of shrimp species in a mixture was difficult due to similar banding patterns between closely related species.     

Fish species identification by fish muscle dry powder as reference material

A method for preparing dry powders from fish muscles is described. The muscle dry powders (MDP) were extracted with low ionic strength buffer and compared with sarcoplasmic extracts of raw muscles by isoelectric focusing on ultra-thin layer (100 μm) polyacrylamide gels. The protein patterns of MDP extracts corresponded to the patterns of the acidic proteins of raw muscle extracts. Because of the species specificity of the patterns, MDP can be used as reference material for fish species identification. The acidic proteins remained extractable after storage of MDP for several months at room temperature.     

Identification of lactic acid bacteria from spontaneous fermentation of 'Almagro' eggplants by SDS-PAGE whole cell protein fingerprinting

In order to complete the previously performed phenotypic identification [Sánchez et al., 2000. Int. J. Food Microbiol. 59 (2000) 9], whole cell protein patterns obtained using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) of 149 lactic acid bacteria (LAB) strains, isolated from 'Almagro' eggplants spontaneous fermentation, were analysed. Phenotypic identification of those strains had revealed the presence of the species Lactobacillus (Lb) plantarum, Lb. pentosus, Lb. brevis biotype 2, Lb. brevis biotype 3, Lb. fermentum, Lactobacillus spp. and Aerococcus viridans. The SDS-PAGE technique generated complex and stable patterns that were easy to interpret and compare with those of the 17 reference strains used in this study. After numerical analysis of the resulting electrophoretic protein patterns, six well-delineated clusters were discriminated that, with some exceptions, correlated well with the different groups phenotypically found. Only two strains showed unique protein patterns without meaningful homology to any of the reference strains used for this study and remained unidentified.     

Quantification of beef myofibrillar proteins by SDS-PAGE

A semi-quantitative determination of beef myofibrillar proteins using sodium, dodecyl sulphate polyacrylamide gel electrophoresis is described. Bovine serum albumin was used as internal standard. Results indicate a linear relationship between densitometric readings after staining with Coomassie brilliant blue R(250) and protein content. Recoveries of four pure proteins, loaded within a range of 0·5-4 μg, were between 92 and 110%. Heating of beef myofibrillar protein samples during dissolution is shown to be unnecessary. The method allows the quantitative study of myofibrillar degradation during beef aging, without overlapping of troponin-T with tropomyosin bands.     

卵白蛋白提取及其可食性膜制备

提取鸡蛋清中的卵白蛋白,制备卵白蛋白粉。采用考马斯亮蓝标准曲线法和凯氏定氮法分析蛋白含量,SDS-PAGE电泳测定纯度。用甘油塑化卵白蛋白,制备可食性蛋白膜。     

Electrophoretic Identification of Fish Species Used in Surimi Products

Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and urea gel isoelectric focusing (IEF) were used to identify species-specific protein bands of raw and cooked fish and surimi samples from Alaska pollock (Theragra chalcogramma) and red hake (Urophycis chuss). In raw samples, species-specific bands were found in the water extracts, while in cooked samples 1% SDS and 8M urea extracts were more effective for species identification in both fish and surimi.     

Species identification of formed fishery products and high pressure-treated fish by electrophoresis: a collaborative study

The suitability and reliability of three electrophoretic methods of fish species identification, urea isoelectric focusing (IEF), sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and native IEF, were evaluated on formed fish fillets and high pressure fish flesh by a collaborative study among four institutes. By following optimized standard operation procedures, the protein patterns of processed fish were compared to patterns of raw reference samples. The method to use depended of the effect of processing on the protein pattern. The proteins obtained from formed products were not denatured and therefore any of the three methods proved to be adequate, with a preference for native IEF which had a better discriminatory power for the species used. The high pressure process altered the proteins, and so only urea IEF and SDS-PAGE methods could be used. For these products, the chosen method should then be the one with the better discriminating power for the species being examined.     

Qualification and quantification of fish protein in prepared surimi crabstick

ABSTRACT:  Species identification and protein quantification in surimi crabstick were achieved using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). When the Lowry and Kjeldahl protein determination methods were compared, the former showed more consistent results. Densitometric scanning of the gels was used for quantification of total fish protein as well as total egg white protein. The lower molecular weight proteins, 30 kDa and lower, proved to be the most useful in fish species identification as well as egg white protein addition. Using a combination of the myosin heavy chain band and the species-specific myosin light chain (Alaska pollock: 22.5 kDa; Pacific whiting: 24.4 kDa) proved the most accurate in calculating fish protein content of the crabstick sample, while for those samples that contained egg white, quantification was accomplished from the densitometric analysis of the overlapping bands of actin (45 kDa) from fish and ovalbumin from egg white. Lysozyme (14.3 kDa) proved to be a unique protein band in determining the presence of egg white when the content of dried egg white was equal to or exceeded 0.5% of the total weight of the final crabstick.