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。     

人脑胶质细胞瘤核染色质非组蛋白的聚丙烯酰胺凝胶电泳分析

用不连续SDS-聚丙烯酰胺凝胶电泳分析了人脑胶质细胞瘤与正常脑细胞核NHCP电泳图谱。从二者的NHCP电泳结果表明,脑胶质细胞瘤增加了一条表观分子量为3万的蛋白区带;表观分子量为1.75万～6.5万的蛋白区带染色明显加深。染色质紫外吸收光谱也有明显差异。总之,脑胶质细胞瘤核NHCP发生质与量的变化。     

五种啮齿类动物核型和血清蛋白质的SDS-聚丙烯酰胺凝胶电泳分析

本文对5种啮齿类动物(岩松鼠、花鼠、五趾跳鼠、棕背(鼠平)和岢岚绒鼠)的核型及其血清蛋白质的SDS-聚丙烯酰胺凝胶电泳进行了分析。通过分析结果,探讨了它们的核型及血清蛋白在其分类中的意义。     

一种简单快速的聚丙烯酰胺凝胶电泳染色法——铜染色法

十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDSPAGE)作为分离鉴定生物大分子的有效手段已广泛用于分子生物学和医学临床工作,迄今为止,用于该技术的染色法多沿用考马斯亮蓝(CBB)和银染色。但上述两法均有其局限性,如蛋白凝胶染色前须经酸醛固定,不易洗脱且操作繁琐,脱色耗时。最近,Lee等报道一种SDS-PAGE负染色法,利用氯化铜分别与凝胶中     

链分离凝胶电泳技术

由于某些原因,(例如,用Maxam—Gibert方法测定序列;与低含量RNA杂交。)有时需要获得双链相互分离的DNA分予片断。通常采用的方法是让变性的DNA在中性琼脂糖凝胶或聚丙烯酰胺凝胶中进行电泳。许多DNA片断的两条互补链在凝胶中有不同的迁移率,其原因尚不清楚。     

妊娠过程中血清蛋白的动态变化

利用聚丙烯酰胺凝胶(PAG)电泳分离技术对266份不同孕期的正常孕妇和妊娠高血压综合征病人的血清蛋白电泳图谱进行了分析。在266份图谱中发现41个染色深度有变化的组分,代表血清蛋白含量的变化。在正常中期妊娠电泳图谱中少数呈1个或2个“V”形异常成分位于P区或O段、M段,其意义尚难肯定。在妊娠高血压综合征病例中没有发现特异性异常蛋白,但血清前白蛋白、白蛋白减少与T-77组分增多和同期正常孕妇相比有显著差异(p<0.05)。     

蛋白质组学实验中聚丙烯酰胺凝胶的考马斯亮蓝染色

在蛋白质组学实验中,蛋白质经聚丙烯酰胺凝胶电泳后的染色是一个十分重要的环节。鉴于其良好的质谱兼容性和操作上的便捷,考马斯亮蓝染色是目前众多实验室中最常用的染色方法。本文介绍了四种各具特色的考马斯亮蓝染色方法。     

多糖和糖蛋白聚丙烯酰胺凝胶电泳染色方法的改进

对多糖聚丙烯酰胺凝胶电泳（PAGE）的缓冲系统进行了改进,尤其是多糖电泳后的染色方法的改进,得出一种操作简便、时间短、实验成本低的PAS染色法。     

一种改进的蛋白质超薄凝胶电泳方法

介绍了一种将聚丙烯酰胺凝胶固定在电泳夹板上的蛋白质电泳方法.通过此方法蛋白质电泳可以在0.4 mm厚的聚丙烯酰胺凝胶上进行.实验证明,经此方法处理的玻板结合凝胶非常牢固,在电泳后的所有处理步骤中都不会发生凝胶脱落现象.     

一种简易的聚丙烯酰胺凝胶干胶制作法

在聚丙烯酰胺凝胶电泳中经常遇到凝胶干胶制作不好的问题,即使在有干胶机的实验室也常发生凝胶裂开、皱缩、电泳条带扩散、干胶质量不理想的情况,一些文献介绍的方法也常发生类似问题。我们在干胶的方法上作了一些摸索和改进,总结出一种简易的、     

Investigation of polyacrylamide hydrogel-based molecularly imprinted polymers using protein gel electrophoresis

In conjunction with polyacrylamide gel electrophoresis (PAGE), molecular imprinting methods have been applied to produce a multilayer mini-slab in order to evaluate how selectively and specifically a hydrogel-based molecularly imprinted polymer (MIP) binds bovine haemoglobin (BHb, ~64.5 kDa). A three-layer mini-slab comprising an upper and lower layer and a MIP, or a non-imprinted control polymer dispersion middle layer has been investigated. The discriminating MIP layer, also based on polyacrylamide, was able to specifically bind BHb molecules in preference to a protein similar in molecular weight such as bovine serum albumin (BSA, ~66 kDa). Protein staining allowed us to visualise the protein retention strength of the MIP layer under the influence of an electric field. This method could be applied to other proteins with implications in effective protein capture, disease diagnostics, and protein analysis.     

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the protein, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.     

Increasing the resolution of polyacrylamide gel electrophoresis by varying the degree of gel crosslinking

Resolution of polyacrylamide gel electrophoresis may be substantially improved by taking advantage of the gel sieving effects of varying concentrations of bisacrylamide crosslinker. A dilution procedure is described which permits simultaneous variation of both total acrylamide concentration and percent crosslinking within a single linear regression analysis.This work was supported by NSF Grant 10584 and NIH Grant 23504.     

Typing and subtyping of haptoglobin from native serum using disc gel electrophoresis in alkaline buffer: application to routine screening

A method with which the six common phenotypes of human haptoglobin can be identified using unseparated serum is described. In contrast to other reported methods, both typing and subtyping of haptoglobin can be performed by polyacrylamide gel electrophoresis in alkaline buffer using 0.1-4.0 microliter of native serum with hemoglobin added. Haptoglobin-hemoglobin complexes are visualized by their peroxidase activity using benzidine and barium peroxide. This relatively inexpensive and fast method seems particularly well suited for the typing and subtyping of haptoglobin from minute amounts in large series of sera and other body fluids and thus may be useful in medical genetics and forensic medicine.     

Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry

Exosomes are 40-100 nm vesicles released by numerous cell types and are thought to have a variety of roles depending on their origin. Exosomes derived from antigen presenting cells have been shown to be capable of initiating immune responses in vivo and eradicating established tumours in murine models. Tumour-derived exosomes can be utilised as a source of tumour antigen for cross-priming to T-cells and are thus of interest for use in anti-tumour immunotherapy. Further exploration into the protein composition of exosomes may increase our understanding of their potential roles in vivo and this study has examined the proteome of exosomes purified from cell supernatants of the melanoma cell lines MeWo and SK-MEL-28. The vesicular nature and size (30-100 nm) of the purified exosomes was confirmed by electron microscopy and sucrose density gradient centrifugation. Western blotting demonstrated the absence of calnexin and cytochrome c, verifying the purity of the exosome preparations, as well as enrichment of MHC class I and the tumour-associated antigens Mart-1 and Mel-CAM. The two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein profiles of exosomes from the two cell lines were highly comparable and strikingly different from the profiles of the total cell lysates. Mass spectrometric sequencing identified proteins present in 49 protein spots in the exosome lysates. Several of these have been identified previously in exosomes but some are novel, including p120 catenin, radixin, and immunoglobulin superfamily member 8 (PGRL). Proteins present in whole-cell lysates that were significantly reduced or excluded from exosomes were also identified and included several mitochondrial and lysosomal proteins, again confirming the proposed endosomal origin of exosomes. This study presents a starting point for future more in-depth protein studies of tumour-derived exosomes which will aid the understanding of their biogenesis and targeting for use in anti-tumour immunotherapy protocols.     

Recovery of proteins on a milligram scale from polyacrylamide electrophoresis gels,exemplified by purification of a retinol-binding protein

An apparatus suitable for the recovery of proteins from polyacrylamide gels on a milligram scale by displacement electrophoresis (isotachophoresis) is described along with a buffer system that is suitable for this purpose with most proteins. The technique is illustrated by the recovery of a protein from a 15% polyacrylamide gel. The recovery was almost quantitative and the eluted protein showed little contamination upon quantitative amino acid analysis and automatic Edman degradation.     

Electrophoresis in the presence of Coomassie brilliant blue R-250 stains polyacrylamide gels during protein fractionation

A method of staining polyacrylamide gels in which the dye is electrophoresed together with the sample is proposed. The method cuts short and simplifies the conventional electrophoresis procedure by eliminating the separate poststaining step. In the gels run in the presence of sodium dodecyl sulfate, the method produces protein staining patterns which are quantitatively identical to the ones obtained by conventional staining procedure. Additional advantages of the method are easy control over the degree of staining and homogenous staining independent of the gel thickness and concentration of the dye.     

Apparatus and method for preparative gel electrophoresis

A new apparatus for preparative gel electrophoresis with continuous elution which includes a miniaturized electrode and elution chamber system is described. The design provides high resolution, high yield, applicability for small and large amounts of peptide material, and easy operation. Furthermore, the apparatus enables a very accurate gel column or gel gradient to be formed. A method for preparative gel electrophoresis in sodium dodecyl sulfate which allows the purification of peptides and proteins without concurrently modifying tryptophane residues or blocking N-terminal α-amino groups is also described.     

Direct chemiluminescent imaging detection of human serum proteins in two-dimensional polyacrylamide gel electrophoresis

A novel chemiluminescence (CL)-based imaging method capable of directly detecting proteins in polyacrylamide gels after electrophoresis is proposed. Human serum proteins are presently detected by a direct CL imaging method after native 2-D PAGE. As a consequence, some proteins, including haptoglobin (Hp), Hp precursor, hemopexin (Hpx) precursor, Ig alpha-1 chain C region, and Complement C3 precursor can be detected and identified by MS and MS/MS techniques. These proteins are all acute phase proteins, which have been defined as biomarkers for certain diseases. Moreover, serum proteins from healthy people and cirrhotic patients were analyzed. A decrease in Hp spots for cirrhotic patients could be confirmed. The CL imaging conditions were optimized, including the concentrations of H(2)O(2) and luminol. The process of CL detection of proteins is simple, and there is no need for specialized equipment. In comparison with the traditional CBB-R250 staining method, the detection sensitivity was improved and the detection period decreased about 70 times. Hence, this technique possesses potentials as a rapid, convenient, and inexpensive analytical technique for protein detection and for the diagnosis of diseases.     

一种改进的双向电泳染色方法

本文涉及了双向电泳过程中的染色方法,即先用考马斯亮蓝染色,将胶上可见蛋白切下再银染的方法。这种方法可最大限度的减少胶中蛋白质点的损失,不仅避免了单一用考马斯亮蓝染色由于灵敏度不高而导致的低丰度蛋白的损失,也避免了单一用银染而使高丰度的蛋白因染色过度导致的损失。同时两种传统的染色方法结合完美,形成的新方法经济实用。