Isoelectric focusing of high-molecular-weight protein complex under native conditions using agarose gel

The isolation and characterization of protein complexes are essential steps toward understanding cellular functions. A method for separating and characterizing high-molecular-weight protein complexes using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with native agarose gel isoelectric focusing (IEF) is described. Using this method, fractions containing high-molecular-weight protein complexes were analyzed. The advantages of using native agarose gel IEF include the ability to concentrate the protein complexes and the ease of handling when performing 2D separations. Although limited with respect to the size of molecules and particles that may be separated, this method is useful for the isolation and characterization of high-molecular-weight protein complexes.     

Separation of the DNA molecules beyond conventional size limits by gel electrophoresis with sodium dodecyl sulfate.

Electrophoretic mobility of DNA through polyacrylamide as well as agarose gels is greatly increased by sodium dodecyl sulfate (SDS). DNA molecules well beyond the conventionally separable size limits are separated readily and rapidly by gel electrophoresis with SDS in a conventional static electric field. Furthermore in optimal concentration gels DNA molecules of similar molecular sizes are separated better from one another in the presence of SDS than without it. Evidence is presented that SDS may act at least in part by altering conformation of DNA. This simple and readily available means for high resolution separation of hitherto impossible sizes of DNA molecules in polyacrylamide and agarose gels in an ordinary static electric field should find general use in molecular genetic analyses. Structural analyses of DNA-protein complexes are also facilitated by virtue of the simultaneous separation of the DNA and protein components on the same gel lane.     

Coupling of native liquid phase isoelectrofocusing and blue native polyacrylamide gel electrophoresis: a potent tool for native membrane multiprotein complex separation

In this study, a new 3D native electrophoretic protocol is proposed for an exhaustive separation and identification of membrane proteins. It is based on native liquid phase isoelectrofocusing (N-LP-IEF) of protein complexes in the first dimension, followed by blue native polyacrylamide gel electrophoresis (BN-PAGE) in the second dimension, where both the pI and the molecular masses of protein complexes (2D N-LP-IEF-BN) were used to separate them in their native form. Finally, each single component can be resolved using denaturing electrophoresis (3D N-LP-IEF-BN-SDS-PAGE). The thylakoid membrane of spinach which contains four big protein complexes was chosen as a model for setting up analytical methods suitable for any membrane proteins. The pI-based MicroRotofor has a number of advantages over BN-PAGE: it does not require the addition of any chemicals, and separation of complexes is based on the protein's real physicochemical properties which inevitably change when dye is added. Results were more easily reproduced than with BN, and the pI of each native complex was also determined. Although some fractions still contained comigrating complexes after MicroRotofor, these were subsequently separated by BN for further analysis. Thus, highly hydrophobic complexes, such as ATP-synthetas and Cyt b6/f, were separated in native form as were various complexes of LHCII trimers, which have different pI but similar molecular masses. SDS-PAGE revealed almost all the subunits from the four photosynthetic complexes, indicating that by using 3D N-LP-IEF-BN-SDS-PAGE it is possible to achieve a greater degree of component identification than with 2D BN-SDS-PAGE.     

Optimization of the scratch assay for in vitro skeletal muscle wound healing analysis

To isolate mitochondrial complexes, we have combined elements from the classic Laemmli protocol and blue native polyacrylamide gel electrophoresis (BN–PAGE) methods to develop a straightforward modified native electrophoresis protocol. This modified protocol presented good resolution for native electrophoresis of inner mitochondrial membrane proteins, where bands were easily visualized with no leftover stain or gel lanes overlap. Enzymatic tests revealed that complexes I and V remain active in the gel. This protocol, designed to overcome specific limitations of the standard protocols, provides a potential methodology to study membrane proteins in their functional form.     

Detection of iron-containing proteins contributing to the cellular labile iron pool by a native electrophoresis metal blotting technique

The labile iron pool (LIP) plays a role in generation of free radicals and is thus the target of chelators used for the treatment of iron overload. We have previously shown that the LIP is bound mostly to high molecular weight carriers (MW>5000). However, the iron does not remain associated with these proteins during native gel electrophoresis. In this study we describe a new method to reconstruct the interaction of iron with iron-binding proteins. Proteins were separated by native gradient polyacrylamide gel electrophoresis and transfered to polyvinilidene difluoride membrane under native conditions. The immobilized iron-binding proteins are then labeled by 59Fe using a 'titrational blotting' technique and visualized by storage phosphorimaging. At least six proteins, in addition to ferritin and transferrin, are specifically labeled in cellular lysates of human erythroleukemic cells. This technique enables separation and detection of iron-binding proteins or other metal-protein complexes under near-physiological conditions and facilitates identification of weak iron-protein complexes. Using a new native metal blotting method, we have confirmed that specific high molecular weight proteins bind the labile iron pool.     

Analysis of 953 Human Proteins from a Mitochondrial HEK293 Fraction by Complexome Profiling

Complexome profiling is a novel technique which uses shotgun proteomics to establish protein migration profiles from fractionated blue native electrophoresis gels. Here we present a dataset of blue native electrophoresis migration profiles for 953 proteins by complexome profiling. By analysis of mitochondrial ribosomal complexes we demonstrate its potential to verify putative protein-protein interactions identified by affinity purification – mass spectrometry studies. Protein complexes were extracted in their native state from a HEK293 mitochondrial fraction and separated by blue native gel electrophoresis. Gel lanes were cut into gel slices of even size and analyzed by shotgun proteomics. Subsequently, the acquired protein migration profiles were analyzed for co-migration via hierarchical cluster analysis. This dataset holds great promise as a comprehensive resource for de novo identification of protein-protein interactions or to underpin and prioritize candidate protein interactions from other studies. To demonstrate the potential use of our dataset we focussed on the mitochondrial translation machinery. Our results show that mitoribosomal complexes can be analyzed by blue native gel electrophoresis, as at least four distinct complexes. Analysis of these complexes confirmed that 24 proteins that had previously been reported to co-purify with mitoribosomes indeed co-migrated with subunits of the mitochondrial ribosome. Co-migration of several proteins involved in biogenesis of inner mitochondrial membrane complexes together with mitoribosomal complexes suggested the possibility of co-translational assembly in human cells. Our data also highlighted a putative ribonucleotide complex that potentially contains MRPL10, MRPL12 and MRPL53 together with LRPPRC and SLIRP.     

High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.     

Identification of heme binding protein complexes in murine erythroleukemic cells: study by a novel two-dimensional native separation -- liquid chromatography and electrophoresis

In the current postgenomic era there is a growing interest in analysis of protein complexes in their native state. Here we present a novel two-dimensional separation technique for assessment of native protein complexes. The method combines native chromatography with native electrophoresis. The approach was used to study heme-binding protein complexes in murine erythroleukemia cells. The cells were metabolically labeled with [(59)Fe]-heme and cellular lysates were separated by anion-exchange chromatography. Fractions containing the (59)Fe isotope were collected, concentrated and further separated by native gel electrophoresis. A total of 13 radioactive protein bands were detected and analyzed by liquid chromatography-tandem mass spectrometry. Thirty-three individual proteins were identified and attributed to four novel multiprotein complexes representing four different 'snapshots' of cellular events involved in hemoglobin biosynthesis.     

Resolution of mitochondrial and chloroplast membrane protein complexes from green leaves of potato on blue-native polyacrylamide gels

Purification of mitochondria and mitochondrial protein complexes from green tissues is often severely impaired by the presence of chloroplasts and their proteins. Here we present a method which allows analysis of respiratory protein complexes from potato leaves. The procedure includes the preparation of an organellar fraction specifically enriched in mitochondria and the separation of organellar protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). For the first time mitochondrial and chloroplast protein complexes have been resolved simultaneously in a native gel. BN-PAGE allowed the separation of eleven bands, including the mitochondrial NADH-dehydrogenase, the bc1 complex and the mitochondrial F1-ATP synthase as well as the chloroplast F1-ATP synthase, the cytochrome b6f complex, the two photosystems and the light harvesting complex. The resolution of the protein complexes in the first dimension was good enough to allow identification of all subunits of individual complexes in the second dimension under denaturing conditions. Thus, BN-PAGE offers an opportunity to analyze mitochondrial and chloroplast protein complexes from a single preparation from very small amounts of tissue. The implications of our findings, for studies on protein expression and turnover in different tissues and developmental stages, are discussed.     

Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE)

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b ₆ f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.     

Native electrophoretic techniques to identify protein–protein interactions

Permanent protein–protein interactions are commonly identified by co‐purification of two or more protein components using techniques like co‐immunoprecipitation, tandem affinity purification and native electrophoresis. Here we focus on blue‐native electrophoresis, clear‐native electrophoresis, high‐resolution clear‐native electrophoresis and associated techniques to identify stable membrane protein complexes and detergent‐labile physiological supercomplexes. Hints for dynamic protein–protein interactions can be obtained using two‐hybrid techniques but not from native electrophoresis and other protein isolation techniques except after covalent cross‐linking of interacting proteins in vivo prior to protein separation.     

Detection of serum proteins by native polyacrylamide gel electrophoresis using Blue Sepharose CL-6B-containing stacking gels

Analysis of serum proteins by native polyacrylamide gel electrophoresis is difficult because albumin is abundant in serum and interferes with the resolution of other proteins, especially alpha-antitrypsin which has mobility that is very similar to that of albumin. We present here a method in which serum proteins are separated by polyacrylamide gel electrophoresis using stacking gels containing Blue Sepharose CL-6B, which has a high affinity for albumin, lipoproteins, kinases, and pyridine-nucleotide-dependent oxidoreductases. During electrophoresis, proteins that bind to Blue Sepharose CL-6B stay in the stacking gel and do not migrate into the separating gel. As a consequence, certain proteins, including alpha(1)-antitrypsin, can be detected as clear bands. This method overcomes the requirement for fractionation of serum samples prior to electrophoresis to remove albumin and allows the simultaneous analysis of many samples.     

The electrophoresis of histones in polyacrylamide gel and their quantitative determination

1. A new method has been devised for the separation of the histone fractions of calf thymus by electrophoresis in polyacrylamide gel at pH2.4. 2. The fractions have been characterized by their relative mobilities with respect to a marker protein, bovine plasma albumin. 3. A method has been developed for the quantitative determination of the separated histone fractions by measuring the colour yields of dye-histone complexes formed in the gel.     

Isolation and crystallization of a unique size category of recombinant Rabies virus Nucleoprotein-RNA rings

In order to study the packaging of rabies virus RNA inside the viral nucleocapsid, rabies nucleoprotein was expressed in insect cells. In the cells, it binds to cellular RNA to form long, helical or short circular complexes, depending on the length of the bound RNA. The circular complexes contained from 9 up to 13 N-protomers per ring. Separation of the rings into defined size classes was impossible through regular column chromatographies or gradient centrifugation. The size classes could be separated by native polyacrylamide gel electrophoresis. A large-scale separation was achieved with a 4% native gel using a preparative electrophoresis apparatus. Crystallization trials were set up with N-RNA rings from three size classes and crystals were obtained in all cases. The best diffracting crystals, diffracting up to 6A, contained rings with 11 N-protomers plus an RNA molecule of 99 nucleotides. The diffraction limit was improved to 3.5A by air dehydration prior to flash freezing.     

Laser‐induced liquid bead ion desorption‐MS of protein complexes from blue‐native gels,a sensitive top‐down proteomic approach

We have developed an experimental approach that combines two powerful methods for proteomic analysis of large membrane protein complexes: blue native electrophoresis (BNE or BN‐PAGE) and laser‐induced liquid bead ion desorption (LILBID) MS. Protein complexes were separated by BNE and eluted from the gel. The masses of the constituents of the multiprotein complexes were obtained by LILBID MS, a detergent‐tolerant method that is especially suitable for the characterisation of membrane proteins. High sensitivity and small sample volumes required for LILBID MS resulted in low demands on sample quantity. Eluate from a single band allowed assessing the mass of an entire multiprotein complex and its subunits. The method was validated with mitochondrial NADH:ubiquinone reductase from Yarrowia lipolytica. For this complex of 947 kDa, typically 30 μg or 32 pmol were sufficient to obtain spectra from which the subunit composition could be analysed. The resolution of this electrophoretic small‐scale approach to the purification of native complexes was improved markedly by further separation on a second dimension of BNE. Starting from a subcellular fraction obtained by differential centrifugation, this allowed the purification and analysis of the constituents of a large multiprotein complex in a single LILBID spectrum.     

A high-definition native polyacrylamide gel electrophoresis system for the analysis of membrane complexes

Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN‐) and high resolution clear native (hrCN‐) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine‐ and deoxycholate‐based native (HDN‐) PAGE. We compared the capacity of HDN‐, BN‐ and hrCN‐PAGE to resolve the well‐studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN‐PAGE. The analysis of isolated chloroplast envelope complexes by HDN‐PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN‐PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.     

茶树叶片蛋白双向电泳体系建立与应用

为开展茶树Camellia sinensis 低温和干旱胁迫下差异蛋白的分离和鉴定,以抗逆性较强的茶树品种‘迎霜’为试材,通过对提取方法、IPG 胶条pH 范围、上样量、分离胶浓度、染色方法的比较,筛选适用于茶树叶片的蛋白质双向电泳体系。结果表明,采用TCA-丙酮法或Tris-HCl 法提取叶片总蛋白,选用17 cm pH 4~7IPG 胶条用于等电聚焦,选择1.6~2.2 mg 上样量、13.5%聚丙烯酰胺凝胶进行分离,随后通过高敏考马斯亮蓝R-250 法染色;最终,叶片各分子量的蛋白充分分离,获得的双向电泳图谱分辨率高、背景清晰、重复性好,适用于‘迎霜’低温和干旱胁迫下叶片差异蛋白分析。     

Coomassie blue-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for direct visualization of polypeptides during electrophoresis

A method for the direct visualization of Coomassie blue-stained polypeptide bands during electrophoresis with subsequent elution of polypeptides and removal of sodium dodecyl sulfate (SDS) and Coomassie blue is described. Primarily it is intended as a means for easy and--because there is no protein fixation step--nearly quantitative recovery of separated polypeptides for amino acid sequencing. It may also be used to obtain rapid information about the protein patterns during a run. Together with our new high resolution SDS-polyacrylamide gel electrophoresis system for small proteins and polypeptides (H. Sch?gger and G. Von Jagow (1987) Anal. Biochem. 166, 368-379) the method described allows the preparative separation of protein fragments as even protein fragments between 1 and 3.5 kDa are easily detected.     

Dodecyl maltoside-sodium dodecyl sulfate two-dimensional polyacrylamide gel electrophoresis of chloroplast thylakoid membrane proteins

A two-dimensional electrophoretic system has been developed for the separation of chloroplast thylakoid membrane proteins. This system incorporates nondenaturing polyacrylamide gel electrophoresis in the presence of the nonionic detergent dodecyl-beta-D-maltoside in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Thylakoid membranes isolated from Spinacia oleracea were solubilized in 1.0% dodecyl-beta-D-maltoside and separated in 4-7% linear acrylamide gradient tube gels which contained 0.05% dodecyl-beta-D-maltoside. After electrophoresis, the tube gels were equilibrated with a sodium dodecyl sulfate-containing equilibration buffer and applied to a 12.5-20% acrylamide linear gradient gel. The Lammelli buffer system was used in both dimensions. The two-dimensional gels were analyzed by staining sequentially with 3,3',5,5'-tetramethylbenzidine-H2O2, Coomassie blue, and silver staining. A number of protein components were identified on "Western blots" of these two-dimensional gels by immunological localization. Membrane protein complexes such as the light-harvesting chlorophyll a/b protein complex, photosystem I, photosystem II, the cytochrome b6/f complex and ribulose bisphosphate carboxylase appear to migrate as essentially intact complexes in the first dimension and appear as vertical series of resolved subunits in the second dimension. This technique complements isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis in providing additional information concerning the subunit composition of membrane protein complexes and may prove to be of general utility for studying the protein composition of other membrane systems.