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1.
目的:探讨Western blot免疫印迹法不同转膜方法和不同抗原抗体比例对磷酸化蛋白表达的检测效果。方法:选择肌球蛋白轻链(myosin light chain,MLC)及其磷酸化蛋白作为研究对象,比较半干转印法、湿转法和1:3000、1:5000、1:10000等抗体稀释比例对磷酸化蛋白检测效果的影响。结果:半干转印法(恒压16V,30 min)观察到蛋白信号断续;而同样样品利用湿转法(恒压130 V,1h)检测发现信号连续且强度明显增高;对于磷酸化蛋白,半干转印法无法观察到磷酸化蛋白信号;而同样样品湿转法检测出现连续信号。统一利用湿转方法进行后续蛋白磷酸化检测,当抗体稀释比为1:3000时,结果出现非特异性条带;降低抗体稀释比为1:5000时无非特异性条带,且蛋白信号效果较好;抗体稀释比为1:10000时条带图像出现弥散且背景较高。结论:选择合适的转膜方式和抗原抗体比例有助于磷酸化蛋白表达检测。  相似文献   

2.
利用三种转移缓冲液,分别以半干和湿转法,检测癌基因产物Bcl-2, 并获得不同强度的印迹结果.不含SDS的转移缓冲液转移效果明显优于含SDS的转移缓冲液,且SDS含量越高印迹带越弱;此外,半干转移与湿转相比,可大大缩短转移时间,但转移效果不及湿转理想且稳定性较差.  相似文献   

3.
蛋白免疫印迹法同时检测大、小分子蛋白的实验条件改进   总被引:1,自引:0,他引:1  
目的:蛋白免疫印迹法是现代生物医学研究中广泛应用于蛋白定性和半定量分析的实验技术。然而,常规采用传统单一浓度凝胶的蛋白免疫印迹法在应用过程中仍有不足之处,如不能同时检测分子量很大和很小的蛋白,因而有必要探索一种增大凝胶有效分离范围的检测方法。本文提出采用组合凝胶来实现更大范围分子量蛋白的同时检测。方法:比较双浓度的组合凝胶与单一浓度凝胶的分离范围以及分析采用组合凝胶,蛋白免疫印迹法对大、小分子蛋白的检测效果。结果:12%/7.5%组合凝胶和15%/7.5%组合凝胶的分离范围显著大于相应的单一浓度凝胶。通过12%/7.5%组合凝胶,蛋白免疫印迹法同时检测到15-300 k Da范围内的大、小分子蛋白。结论:组合凝胶有助于蛋白免疫印迹法对分子量相差很大的蛋白进行同时检测分析,具有很强的实用性。  相似文献   

4.
自制显示系统检测免疫印迹中的目的蛋白   总被引:1,自引:0,他引:1  
目前蛋白免疫印迹显示系统有显色法和化学发光法,因化学发光法具有灵敏、快速、准确等特点而倍受科研工作者的青睐,但一般商品化的化学发光试剂盒价格颇为昂贵,为此,在参考文献的基础上作了一些改进,将Tris Cl的pH值从7.5提高到11.0,同时提高了对碘苯酚的浓度(从1mmol/L提高到2mmol/L),得到了自制的化学发光试剂。首先用其检测了大肠杆菌中表达的2种不同分子量的蛋白(gam,13kDa;bet,30kDa),发现当蛋白上样量为2~20ng时,各蛋白条带均清晰显示。随后的斑点免疫印迹实验表明:自制及进口化学发光试剂在检测酶标二抗效价方面有着同样高的敏感性。  相似文献   

5.
登革2型病毒E蛋白在酵母菌中的分泌表达   总被引:5,自引:0,他引:5  
以pPICZ α B为载体,应用RT-PCR从感染D2V的C6/36病变细胞中克隆全长E基因,电转 化法将重组质粒整合入巴斯德毕赤氏酵母菌,经抗生素筛选、表型鉴定和PCR分析得到Mut+型的多拷贝整合菌,经甲醇诱导培养可产生69kD的融合蛋白,与含组氨酸尾的D2V包膜糖 蛋白分子量理论值相符;免疫印迹证实该表达产物可与D2V E特异性单抗和D2V多抗进行反应; 表达产物经金属螯合亲和层析可获得纯化的含组氨酸尾的E融合蛋白并保留其免疫反应性. 研究显示克隆的全长D2V E基因可在毕赤氏酵母菌中高效分泌表达,E融合蛋白最大表达量0.1g/L.  相似文献   

6.
目的:纯化人vasorin(VASN)蛋白胞外结构域的单克隆抗体并鉴定。方法:用表达人VASN蛋白胞外结构域单克隆抗体的杂交瘤细胞免疫小鼠,收集腹水,纯化抗体;ELISA检测单抗的特异性和亲和力,Western印迹、免疫共沉淀、细胞免疫荧光等实验检测单抗的特异性与可能的用途。结果:纯化获得2株抗VASN胞外结构域单抗V20和V21,ELISA结果显示二者均特异性强、亲和力高;Western印迹显示2株单抗均可结合Hep G2细胞中的VASN蛋白,以V20为佳;免疫共沉淀实验结果显示V21能够钓取Hep G2细胞中的VASN蛋白及细胞培养上清中的分泌型VASN;免疫荧光实验结果显示V21能与Hep G2细胞的VASN蛋白结合。结论:纯化获得2株抗人VASN胞外结构域单抗,为进一步研究VASN蛋白的生物学功能提供了实验工具。  相似文献   

7.
[目的]基于改良的对比蛋白提取结果的方法,探讨两种不同总蛋白提取方法对不同丰度蛋白提取得率的影响。[方法]小胶质细胞株N9总蛋白及商品化预染蛋白标准品,行聚丙烯酰胺凝胶电泳(SDS-PAGE),三种常用图像软件定量免疫印迹结果,比较线性相关系数R~2;选择R~2值最高的软件,灰密度定量不同丰度p-IκBα经RIPA裂解液法和Minute试剂盒法蛋白提取后免疫印迹检测的p-IκBα蛋白得率。[结果]三种软件定量分析同一免疫印迹结果显示,Image Studio Lite的上样量与条带灰密度值的R~2最大(R~2=0.998);运用Image Studio Lite定量免疫印迹检测p-IκBα高丰度组,发现使用RIPA裂解液法比Minute法的p-IκBα提取得率高出7.5%,而p-IκBα低丰度组,前者p-IκBα的得率只有后者的41%,差异显著(P0.05)。[结论]Minute试剂盒法在免疫印迹检测中对低丰度蛋白具有高提取得率的优越性。  相似文献   

8.
为建立一种快速筛选酵母高表达菌株的方法,将生长在固体培养基上的新转化的表达菌落原位转移到醋酸纤维素薄膜上,再利用硝酸纤维素薄膜原位捕获醋酸纤维素薄膜上的蛋白,然后用免疫印迹的方法检测原位转印到硝酸纤维素膜上菌落分泌蛋白,根据免疫印迹的强弱进行高表达菌株的筛选。结果显示,经过菌落原位转印和免疫印迹显色,显色信号强的菌落表达量相对较高。因此,双膜法原位检测是一种快速、高效的筛选酵母高表达菌株的方法。  相似文献   

9.
目的研制青霉素结合蛋白2a(PBP2a)单克隆抗体(Mc Ab),为建立耐甲氧西林金黄色葡萄球菌(MRSA)免疫层析检测方法提供检测用抗体。方法以基因工程抗原r PBP2a免疫BALb/c小鼠,通过常规小鼠B淋巴细胞杂交瘤技术制备单克隆抗体,采用免疫印迹技术(Western blotting)分析单克隆抗体特异性。选取敏感、特异的单克隆抗体进行金标记和硝酸纤维膜包被,建立胶体金免疫层析检测方法。结果共获得11株分泌抗r PBP2a的杂交瘤细胞,其中6株分泌的单克隆抗体能够与天然PBP2a呈阳性反应。用其中2株单克隆抗体建立的PBP2a胶体金免疫层析方法,可在5~20 min内完成检测。结论获得了特异性针对PBP2a蛋白的单克隆抗体,并初步建立了检测PBP2a蛋白的胶体金免疫层析检测方法,为临床快速、简便检测产生PBP2a的细菌提供了检测方法。  相似文献   

10.
孙建和  蒋静  陆苹 《中国病毒学》2004,19(3):232-236
将IBDV上海超强毒株的多聚蛋白基因(vp2-4-3)克隆入真核表达载体pALTER-MAX,构建成功pALTER-MAX-VP2-4-3真核表达质粒,经纯化后,pALTER-MAX-VP2-4-3在LipofectamieTM 2000介导下转染Vero细胞、11日龄鸡胚的绒毛尿囊膜(CAM)和肌肉注射2日龄的雏鸡,1周后,分别提取细胞或组织中的总DNA或总RNA,用DIG标记探针均可检测到阳性杂交信号;转染的Vero细胞飞片和肌肉冰冻切片,进行免疫荧光检测均呈现阳性结果;转染的鸡胚CAM匀浆上清,用兔抗IBDV超强毒的高免血清,经Dot-ELISA检测呈现阳性.表明转染后基因获得表达,表达的蛋白具有免疫反应性.  相似文献   

11.
The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a "black box" because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as β-actin, β-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative and reliable.  相似文献   

12.
Western blot transfer buffer was modified to substitute the acute poison methanol, with the common rubbing alcohol, isopropanol in concentrations of as low as 5 % for protein electrotransfer. Commercially available molecular weight markers and rabbit serum were run on polyacrylamide gels and shown to be transferred adequately to both nitrocellulose and polyvinylidene difluoride membranes under either wet or semi-dry conditions with similar results in all cases. This procedure was successfully used for immunodetection of the rabbit IgG heavy chain from serum. Therefore, this represents a good alternative for less toxic and environmentally friendly conditions for western immunoblotting of proteins.  相似文献   

13.
A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.  相似文献   

14.
The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.  相似文献   

15.
We describe here the use of Alta, a pre-existing scarlet-red stain of cosmetic use, for staining proteins on sodium dodecyl sulfate (SDS) polyacrylamide gels, as well as for a single step staining of gels and nitrocellulose membranes during Western blot analysis. This stain, which is composed of 0.8% Crocein scarlet (brilliant crocein) and 0.2% Rhodamine B, is inexpensive, easy to use and nearly as sensitive as Coomassie Brilliant Blue (CBB) R-250. The gels can be stained in 10% Alta (2 h) and can be destained effectively only with 7% acetic acid as opposed to the conventional destainer (methanol/acetic acid/water) required for CBB-stained gels. In an alternative procedure, the proteins can be stained on the gel while electrophoresis by simply using 5% Alta in the top tank buffer and the stain can be viewed under UV-transilluminator. This procedure can also be used for Western blot analysis, as a single step procedure for staining of proteins on the gel as well as on the nitrocellulose membrane, as the stain is retained on the membrane after protein transfer. Thus, this staining procedure allows monitoring of proteins after each step in the Western blot, thereby eliminating the need to run separate gels for staining and Western blot analysis, and also the need for Ponceau Red S staining of the nitrocellulose membrane during Western blot analysis.  相似文献   

16.
A method for the reproducible and quantitative electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a single sheet of either Zetabind or Gene Screen Plus membranes is presented. This procedure uses commercially available equipment and includes three crucial parameters: the omission of methanol from the transfer buffer, the use of thin (0.75-mm) resolving gels, and a newly developed protocol for pretreatment of the polyacrylamide gel after electrophoresis and before electroblotting. This combination of parameters yields a blot that both qualitatively and quantitatively reflects the proteins in the original polyacrylamide gel.  相似文献   

17.
We have developed a method to transfer proteins from a silver-stained polyacrylamide gel to a polyvinylidene difluoride (Immobilon-P) transfer membrane (Millipore, Bedford, MA). If the silver stained gels are rinsed in 2 x SDS Laemmli sample buffer prior to transfer, almost all proteins can be transferred comparably to non-stained controls. Some proteins stained with silver can be directly transfer, almost all proteins can be transferred comparably to non-stained controls. Some proteins stained with silver can be directly transferred to a single sheet of Immobilon-P without a prior rinse in sample buffer. Most important in the Western blot the antigenicity of the transferred protein is retained in either way. The method described is simple, inexpensive and versatile. A slight modification of the technique permits one to extract minor proteins, or detect their antigenic activities, without contamination of contiguous proteins.  相似文献   

18.
Two-dimensional gel electrophoresis (2DE) and SDS-PAGE are the two most useful methods in protein separation. Proteins separated by 2DE or SDS-PAGE are usually transferred to membranes using a variety of methods, such as electrophoretic transfer, heat-mediated transfer, or nonelectrophoretic transfer, for specific protein detection and/or analysis. In a recent study, Pettegrew et al.1 claim to reuse transfer buffer containing methanol for at least five times for transferring proteins from SDS-PAGE to polyvinylidene difluoride. They add 150–200 ml fresh transfer solution each time for extended use as a result of loss of transfer buffer. Finally, they test efficiency of each protein transfer by chemiluminescence detection. Here, we comment on this report, as we believe this method is not accurate and useful for protein analysis, and it can cause background binding as well as inaccurate protein analysis.  相似文献   

19.
We have developed conditions for the efficient electrotransfer from polyacrylamide gels to nitrocellulose sheets of a broad size range of proteins (Mr 8,000 to Mr greater than 400,000). The important features of this procedure include a two-step electrotransfer, beginning with elution of low-molecular-weight polypeptides at a low current density (approximately 1 mA/cm2) for 1 h, followed by prolonged electrotransfer (16-20 h) at high current density (approximately 3.5-7.5 mA/cm2) in conditions that favor the elution of high-molecular-weight proteins. The transfer buffer includes 0.01% sodium dodecyl sulfate to enhance protein elution, and 20% methanol to improve the retention of proteins on the nitrocellulose sheet. The nitrocellulose is air-dried after transfer is complete to eliminate loss of proteins during subsequent processing. This transfer procedure works well with proteins prepared from many different cell types, and is suitable for use with all polyacrylamide gel systems tested. With little or no modification, our method should also be applicable to transfer membranes other than nitrocellulose.  相似文献   

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