一种小麦高、低分子量麦谷蛋白亚基的提取方法

用7.5%的异丙醇和0.3 mol/L的NaI去除醇溶蛋白和其他单体蛋白, 以二硫苏糖醇(DTT)为强还原剂, 以4-乙烯基吡啶 (VP)保护巯基, 防止其重新氧化。在25%的异丙醇和0.04 mol/L的Tris-HCl (pH=8.0)缓冲液中提取小麦总麦谷蛋白亚基、在4%浓缩胶和13%分离胶的不连续分离体系中进行SDS-PAGE电泳, 结果表明, 该方法不仅能有效去除醇溶蛋白和其他蛋白对麦谷蛋白亚基电泳的影响, 且高分子量麦谷蛋白亚基 (HMW-GS) 和低分子量麦谷蛋白亚基 (LMW-GS)的提取分离一步完成, 更重要的是, 利用该方法提取出的HMW-GS和LMW-GS在电泳分析中, 具有高的分辨率, 可以有效区分各电泳谱带, 为进一步研究奠定了基础。     

棉铃虫围食膜蛋白的电泳分离技术

比较了棉铃虫Helicoverpa armigera(Hübner)围食膜不同处理方法,不同围食膜数量,在不同电泳下的分离效果,筛选最佳的围食膜蛋白分离技术。结果表明:冷冻干燥与非冷冻干燥处理围食膜后进行SDS-PAGE电泳分离效果基本相同,建议使用冷冻干燥后处理较好。取1、3、5条围食膜进行SDS-PAGE电泳,都能将围食膜蛋白分离,且分离效果基本一致,建议5条围食膜最合适,具有可靠性和代表性。浓缩胶为5%、分离胶分别为8%、10%和12%时棉铃虫围食膜的SDS-PAGE电泳分离效果差异不大,而分离胶为12%时效果较好。无水三氟利克酸处理围食膜后进行NuPAGE电泳,分离出的围食膜蛋白大约30多种,远远超过上述方法分离的16²0种,且需要的围食膜材料少,分离效果最佳,但费用高。     

不经染色直接从SDS-PAGE制备凝胶中准确切下所需蛋白条带的方法

蛋白样品的垂直板SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE)不但是一种最常用的蛋白分析方法,也经常用于蛋白质的制备。从电泳凝胶上纯化蛋白,一般都要先用考马斯亮蓝染色,然后切下所需的蛋白条带。这里介绍一种可以不染色,直接从SDS-PAGE制备凝胶上准确切割所需蛋白条带的方法。与染色后切胶的方法相比,这种方法简单、省时,分离到的蛋白容易从胶中洗脱回收,并可明显提高回收率,而且省去了令人烦怖的从回收的蛋白中脱去染色时结合的染料的问题。作者曾用此方法分离过多种蛋白,屡试不爽。这种方法与一般的SDS-PAGE制备电泳的差别主要在电泳结束后对凝胶的处理上。     

用于荧光辅助糖电泳寡糖标尺的构建

荧光辅助糖电泳(FACE)是一种简洁廉价的分离糖类方法。寡糖首先与8-氨基萘基-1,3,6-三磺酸(ANTS)反应标记,然后,ANTS标记的寡糖通过在32％丙烯酰胺-2．4％双丙烯酰胺组成的分离胶上电泳从而得以相互分离。结果表明,电泳图谱能准确反映寡糖的聚合度梯度,因而,一种具有连续聚合度的淀粉水解液的荧光标记电泳图谱可以作为荧光辅助糖电泳的分子量标尺。     

两种酶谱方法在κ-卡拉胶酶活性鉴定中的应用

对分离纯化后的κ-卡拉胶酶进行SDS-PAGE电泳和酶谱试验鉴定,比较κ-卡拉胶酶活性鉴定中的两种酶谱试验方法。一种是先进行非变性PAGE电泳,电泳完毕,将电泳胶与事先准备好的底物胶叠合在一起,35℃孵育液孵育。另一种是在此试验方法基础上进行改进,直接在电泳分离胶中加入0.2%底物,进行SDS-PAGE电泳,电泳结束,用TritonX-100将电泳胶复性,孵育液孵育。试验结果表明改进后的酶谱方法操作简单,具有良好的灵敏度和精确的定位。同时,利用改进后的酶谱方法对κ-卡拉胶酶活性进行了反应时间的研究,结果显示,反应时间为8 h时,降解条带最清晰,最有利于相似分子量酶的辨别。     

小麦叶片类囊体膜蛋白复合体的分离和分析技术研究

膜蛋白复合体在生命活动中执行着重要的生物化学功能,具有重要的研究意义,但由于其低表达和高度疏水的特点,完整状态和低丰度的膜蛋白复合体的分离分析仍是一大挑战.首先,采用蓝绿温和胶非变性电泳作为第一维分离技术,大量分离制备叶绿体类囊体膜蛋白复合物;其次,采用电洗脱方法差异富集第一维电泳分离到的各种蛋白复合体;最后,采用SDS-PAGE作为第二维分离技术,分析差异富集的不同种蛋白复合体的亚基构成.研究结果表明,利用这种分离分析模式,能有效地富集膜蛋白复合体尤其是低丰度膜蛋白复合体,明显提高第二维分离分析的分辨率和蛋白覆盖率.     

小分子肽的Tricine-SDS-PAGE分离方法

在蛋白质的生化分析和基因表达产物的分离纯化中 ,经常要把某种或某些蛋白质成分分离开来。聚丙烯酰胺凝胶电泳 ( SDS- PAGE)是分离蛋白质的常用生化方法 ,样品的蛋白质分子热变性解聚后与 SDS结合形成带负电的蛋白质 - SDS复合物 ,复合物在电泳中的迁移率取决于蛋白质的分子大小 ,使用均匀浓度的 SDS-PAGE来分析分子量在 15～ 2 0 0 k D的蛋白质时 ,电泳迁移率与分子量的对数成线性关系。但常规定 Tris-甘氨酸 -盐酸系统中电泳分离分子量小于 10 k D的多肽效果差。作者在分离小分子肽的实验中改进了一套较简便的分离小分子肽的 T…     

两种电泳系统分离裙带菜色素-蛋白复合物的比较

裙带菜的类囊体膜经过去污剂癸基-N-甲基匍萄糖胺增溶,采用SDS-PAGE分离技术,在Tris-Gly和Tris-硼酸两种电泳系统中分离其色素-蛋白质复合物,并比较其复合物的光谱特性。结果表明:采用Tris-Gly电泳分离系统从裙带菜中分离到8种色食-蛋白质复合物,分别是CP Ia、CPI、LHC₁、CPa、LHC₂、LHC₃、LHC₄和LHC₅。在Tris-硼酸电泳分离系统中共分离到5种色素-蛋白质复合物,分别是CPI、CPa、LHC₁、LHC₂、LHC₃。吸收光谱和荧光光谱的测定结果表明,两种电泳系统中分离的相对应条带的光谱特性基本相近。     

天花粉蛋白胰酶肽图谱的毛细管区带电泳法分析

目的:以天花粉蛋白胰蛋白酶解肽段为测定对象,用毛细管区带电泳法(CZE)研究天花粉蛋白的肽图谱分离条件。方法:采用未涂层石英毛细管(长50cm,内径75μm,有效长度42cm),以50mmol/L磷酸盐和150mmol/L三氟乙酸溶液为运行缓冲液,在25℃、pH2.0和压力为3447.4Pa(×10s)的条件下进样,以12kV恒压电泳分离,检测波长214nm。结果:运用CZE也能较好地对天花粉蛋白进行肽图谱分离,在缓冲体系中加入离子对试剂三氟乙酸,可极大地改善多肽的峰形和分辨率;同时运用反相高效液相色谱(RP-HPLC)技术,也很好地鉴定了部分肽段在CZE和RP-HPLC肽图谱中的对应关系。结论:与传统的RP-HPLC分析天然或重组蛋白肽图谱相比,CZE也不失为一种鉴定蛋白肽图谱的有效、快速和简单的方法。     

转化法分离枯草芽孢杆菌8a5α-淀粉酶基因

用EcoRI部分降解枯草芽孢杜菌8a5染色体DNA,琼脂糖疑胶电泳分离纯化各降解片段,用转化法逐一测定各降解片段的转化活性。实验结果证明,a-淀粉酶基因本身可作为选择性标记用于a-淀粉酶基因的分离。用Hind III降解的λ-DNA 作为分子量对照,确定能高效转化淀粉酶基因的DNA片段大小约4.3kb.本实验获得的a-淀粉酶基因的转化率为1×10⁴转化子/μg DNA.     

SDS聚丙烯酰胺凝胶电泳快速染色新方法的研究

通过几种金融盐溶液对SDS聚丙烯酰胺凝胶电泳染色的实验表明,0.25mol/L的CaCl2和MgCl2溶液能够对蛋白质进行有效的染色,经这2种溶液染色的蛋白质都能够从凝胶中洗脱回收。尤其是CaCl2法灵敏度更高,而且蛋白质条带形成之后也十分稳定,所以在运用制备电泳纯化蛋白质时这种新的染色方法较适用。     

Pulse electrophoresis of muscle myosin heavy chains in sodium dodecyl sulfate-polyacrylamide gels

We have developed a new method that provides enhanced resolution of myosin heavy chain (MHC) isoforms by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The key feature of this protocol involves the application of current to slab SDS gels in a pulsatile, repetitive manner rather than continuously as in standard gel systems. This protocol, designated pulse electrophoresis, was achieved by means of a device that intermittently gates the output of a conventional power supply. When used in long (32 cm) separating gels, pulse electrophoresis not only significantly improves the resolution of MHC isoforms compared to conventional systems, but also reduces common artifacts associated with long running times, such as blurred bands and comingling of closely spaced bands. In addition to the increased resolution of protein bands, pulse electrophoresis also allows detection of bands corresponding to previously unidentified MHC isoforms in mammalian and avian tissue. In rat myocardium, for example, pulse electrophoresis revealed three MHC isoform bands, two of which appeared to correspond to two alpha-MHC subspecies. Alternative splicing of the rat alpha-MHC gene is known to generate two isoform species differing by inclusion (or exclusion) of a single glutamine residue, whose relative levels of expression correspond nicely with the amounts of each band identified in this study. Therefore, we cannot rule out that the system presented here may be sufficiently sensitive to differentiate between high molecular weight proteins differing in a single amino acid.     

东亚飞蝗主要过敏原的分析、鉴定与纯化

通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离东亚飞蝗Locusta migratoria manilensis(Meyen)的蛋白质组分并测定其分子量,收集过敏病人血清,采用免疫印迹(Western—blotting)法鉴定其过敏原成分,通过凝胶过滤层析对东亚飞蝗过敏原进行分离纯化。结果表明:东亚飞蝗蛋白粗提液条带大概有30条左右,其中主带大约有10条,相对分子量约为13、15、25、28、40、45、55、70、100、110ku,其中蛋白含量最丰富的约在70ku左右。免疫印迹结果显示,蝗虫过敏条带主要有5条,相对分子量分别约为19、29、38、70、130ku。通过凝胶过滤层析对东亚飞蝗过敏原进行分离纯化,得到了一个高纯度相对分子质量约为70ku东亚飞蝗过敏原,并且发现了一个相对分子质量约为130ku的蝗虫新过敏原。本研究为临床上蝗虫食物变态反应性疾病的诊断和治疗奠定基础。     

Renaturation of Leishmania donovani 3'-nucleotidase following sodium dodecyl sulfate-polyacrylamide gel electrophoresis

1. Renaturation of a 3'-nucleotidase from the surface membrane of Leishmania donovani promastigotes was achieved following polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). 2. Enzyme activity was detected in situ in gels, following SDS removal, by incubating the gels in reaction mixtures containing 3'-AMP or 3'-UMP as substrate followed by staining for the inorganic phosphate (Pi) reaction product with malachite green-molybic acid solution. 3. Conditions for the removal of SDS by diffusion and for the renaturation of enzyme activity are described including evidence for the detergent requirement, which is best satisfied by 3[(3-cholamidopropyl)-dimethylammonio]2-hydroxy-1-propane sulfonate (CHAPSO). 4. Results indicate that the 3'-nucleotidase migrates under these conditions as a polypeptide with an Mr of 43,000.     

Neonatal myosin heavy chains are not expressed in Ni-induced rat rhabdomyosarcoma

Myosin heavy chain (MHC) composition of chemically-induced rhabdomyosarcoma (RMS) was analyzed by gel electrophoresis and Western blotting using a panel of monoclonal antimyosin antibodies specific for embryonic-, neonatal-, slow- and adult fast-type MHC isoforms. Myosin extracted from tumours and electrophoresed on 6%-sodium dodecyl sulfate (SDS)glycerol gels was found to migrate as three distinct MHC components. These polypeptides were present in different relative amounts in the five RMS studied. Western blotting experiments revealed that variable proportions of embryonic-, slow- and adult fast-, but not neonatal-type, MHC isoforms are consistently expressed in RMS. Indirect and double immunofluorescence procedures applied to cryosections of tumoral tissue showed that: (a) RMS cells were unreactive with antineonatal-type-MHC antibody, (b) the majority of neoplastic, desmin-positive, cells contained embryonic- as well as adult fast-type MHCs and (c) a minority of cells were labelled by anti-slow MHC antibody. The results of this study indicate that there is no obligatory sequence of MHC isoform expression in the molecular transition (emb----neo----adult) which occurs during rat skeletal myogenesis.     

Isoforms of wild type proteins often appear as low molecular weight bands on SDS‐PAGE

Immunoblotting, after polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS‐PAGE), is a technique commonly used to detect specific proteins. SDS‐PAGE often results in the visualization of protein band(s) in addition to the one expected based on the theoretical molecular mass (TMM) of the protein of interest. To determine the likelihood of additional band(s) being nonspecific, we used liquid chromatography – mass spectrometry to identify proteins that were extracted from bands with the apparent molecular mass (MM) of 40 and 26 kD, originating from protein extracts derived from non‐malignant HEK293 and cancerous MDA‐MB231 (MB231) cells separated using SDS‐PAGE. In total, approximately 57% and 21% of the MS/MS spectra were annotated as peptides in the two cell samples, respectively. Moreover, approximately 24% and 36.2% of the identified proteins from HEK293 and MB231 cells matched their TMMs. Of the identified proteins, 8% from HEK293 and 26% from MB231 had apparent MMs that were larger than predicted, and 67% from HEK293 and 37% from MB231 exhibited smaller MM values than predicted. These revelations suggest that interpretation of the positive bands of immunoblots should be conducted with caution. This study also shows that protein identification performed by mass spectrometry on bands excised from SDS‐PAGE gels could make valuable contributions to the identification of cancer biomarkers, and to cancer‐therapy studies.     

Isoform diversity of giant proteins in relation to passive and active contractile properties of rabbit skeletal muscles

The active and passive contractile performance of skeletal muscle fibers largely depends on the myosin heavy chain (MHC) isoform and the stiffness of the titin spring, respectively. Open questions concern the relationship between titin-based stiffness and active contractile parameters, and titin's importance for total passive muscle stiffness. Here, a large set of adult rabbit muscles (n = 37) was studied for titin size diversity, passive mechanical properties, and possible correlations with the fiber/MHC composition. Titin isoform analyses showed sizes between approximately 3300 and 3700 kD; 31 muscles contained a single isoform, six muscles coexpressed two isoforms, including the psoas, where individual fibers expressed similar isoform ratios of 30:70 (3.4:3.3 MD). Gel electrophoresis and Western blotting of two other giant muscle proteins, nebulin and obscurin, demonstrated muscle type-dependent size differences of < or =70 kD. Single fiber and single myofibril mechanics performed on a subset of muscles showed inverse relationships between titin size and titin-borne tension. Force measurements on muscle strips suggested that titin-based stiffness is not correlated with total passive stiffness, which is largely determined also by extramyofibrillar structures, particularly collagen. Some muscles have low titin-based stiffness but high total passive stiffness, whereas the opposite is true for other muscles. Plots of titin size versus percentage of fiber type or MHC isoform (I-IIB-IIA-IID) determined by myofibrillar ATPase staining and gel electrophoresis revealed modest correlations with the type I fiber and MHC-I proportions. No relationships were found with the proportions of the different type II fiber/MHC-II subtypes. Titin-based stiffness decreased with the slow fiber/MHC percentage, whereas neither extramyofibrillar nor total passive stiffness depended on the fiber/MHC composition. In conclusion, a low correlation exists between the active and passive mechanical properties of skeletal muscle fibers. Slow muscles usually express long titin(s), predominantly fast muscles can express either short or long titin(s), giving rise to low titin-based stiffness in slow muscles and highly variable stiffness in fast muscles. Titin contributes substantially to total passive stiffness, but this contribution varies greatly among muscles.     

SDS microslab linear gradient polyacrylamide gel electrophoresis

An improved sodium dodecyl sulfate (SDS) microslab linear gradient polyacrylamide gel electrophoresis (PAGE) technique has been developed. Several important features present in this microslab SDS-PAGE system include (1) high resolution and sensitivity; (2) rapid electrophoresis, staining, and destaining; (3) high reproducibility; and (4) low cost of construction and operation. Several gels are east at once between unmodified commercially available microslides separated by 0.5-mm thick Teflon spacers. The total time from start of electrophoresis to completion of destaining spans 2 hr. Gels are dried between transparent cellophane membranes in 1 hr and can be easily scanned with a microdensitometer. As little as 20 ng of a purified protein stained with Coomassie blue is detectable.     

Detection of fluorescence dye-labeled proteins in 2-D gels using an Arthur 1442 Multiwavelength Fluoroimager

Labeling of proteins with SYPRO Orange, SYPRO Red, and SYPRO Ruby after 2-D polyacrylamide gel electrophoresis (PAGE) using plastic-backed immobilized pH gradient (IPG) strips and precast SDS polyacrylamide gels was tested. Protein spots were detected using an Arthur 1442 Multiwavelength Fluoroimager. The labeling methods described allow detection of proteins both after isoelectric focusing (IEF) and PAGE with a sensitivity higher than or comparable to standard silver staining methods. In addition to the post-labeling methods mentioned above, pre-labeling with the cysteine-specific fluorophore monobromobimane before 2-D PAGE is a sensitive, fast, and cost-effective alternative to existing staining protocols.     

On the titin isoforms

By our modified SDS gel electrophoresis and immunoblotting, the isoform composition of titin in skeletal and cardiac muscles of human and animals was studied to reveal new titin forms above 3700 kDa in size. The data obtained suggest that the new large-size titin species are the intact (original) isoforms of this protein, whereas the known N2A, N2B, and N2BA titin bands in electrophoregrams correspond to their fragments.