Advantages and limitations of clear-native PAGE

Clear-native PAGE (CN-PAGE) separates acidic water-soluble and membrane proteins (pI < 7) in an acrylamide gradient gel, and usually has lower resolution than blue-native PAGE (BN-PAGE). The migration distance depends on the protein intrinsic charge, and on the pore size of the gradient gel. This complicates estimation of native masses and oligomerization states when compared to BN-PAGE, which uses negatively charged protein-bound Coomassie-dye to impose a charge shift on the proteins. Therefore, BN-PAGE rather than CN-PAGE is commonly used for standard analyses. However, CN-PAGE offers advantages whenever Coomassie-dye interferes with techniques required to further analyze the native complexes, e.g., determination of catalytic activities, as shown here for mitochondrial ATP synthase, or efficient microscale separation of membrane protein complexes for fluorescence resonance energy transfer (FRET) analyses. CN-PAGE is milder than BN-PAGE. Especially the combination of digitonin and CN-PAGE can retain labile supramolecular assemblies of membrane protein complexes that are dissociated under the conditions of BN-PAGE. Enzymatically active oligomeric states of mitochondrial ATP synthase previously not detected using BN-PAGE were identified by CN-PAGE.     

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of cytoplasmatic protein complexes from Chlorobium tepidum

Chl. tepidum is a Gram-negative green-sulfur bacterium, which is strict by anaerobic and grows by utilizing sulfide or thiosulfate as an electron source. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes. In particular, the Chl. tepidum-soluble proteome was monitored under native condition by using BN-PAGE. The BN-PAGE protein complexes map was analyzed by MALDI-TOF MS after trypsin treatment and from 42 BN proteins bands, 62 different proteins were identified. Additionally, functional information regarding protein–protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. One-hundred and seventy gel bands were spotted, out of which 187 different proteins were identified. The identified proteins belong to various functional categories like energy metabolism, protein synthesis, amino acid biosynthesis, central intermediate metabolism, and biosynthesis of cofactors indicating the potential of the method for elucidation of functional proteomes.     

Solubilization of membrane protein complexes for blue native PAGE

Blue native PAGE is an electrophoretic technique for high-resolution separation of membrane proteins. The method has been proven especially useful for investigation of native protein complexes enabling a characterization of potential protein-protein interactions in the context of functional proteomics. Blue native PAGE is easy to realise, results are reproducible and a high number of protocols are available. However, care should be taken during solubilization of protein complexes to achieve significant results in BN-PAGE analysis. Solubilization of membranes and proteins is not only influenced by detergent-lipid and detergent-protein interactions but also by lipid-lipid, lipid-protein and protein-protein interactions. Interactions have been investigated experimentally and theoretically. But, in practice, the experimental results do not always mirror the theoretical basis and therefore optimal solubilization conditions for each membrane and membrane protein complex should be investigated individually to tap the full potential of BN-PAGE analysis.     

Identification of cytoplasmic and membrane-associated complexes in human embryonic stem cells using blue native PAGE

Human embryonic stem cells (hESCs) have great potential for use in developmental biology studies, functional genomics applications, drug screening, and regenerative medicine. A detailed understanding of the molecular mechanisms that are responsible for maintaining the undifferentiated and pluripotent nature of hESCs is essential for their effective therapeutic application. It has become evident that many complex cellular processes are carried out by assemblies of protein molecules (protein complexes). Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to separate protein complexes from whole cell lysates. Using BN-PAGE, we resolved cytoplasmic and membrane-associated complexes from hESCs and characterised their composition, stoichiometry, and dynamics by denaturing SDS-PAGE. The reliability of the fractionation was examined by western blot analysis of membrane and cytosolic markers. MALDI TOF/TOF mass spectrometry identified 119 cytosolic and 69 membrane proteins from the BN-PAGE proteome maps. Potential protein complexes were validated by computational prediction of possible protein-protein interactions using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. Based on BN-PAGE gels and validation by databases, 82 heteromultimeric and 47 homomultimeric protein complexes have been found in hESCs. Resolving some of the protein complexes provided insight into the function of previously uncharacterised complexes in hESCs.     

Blue Native electrophoresis to study mitochondrial and other protein complexes

The biogenesis and maintenance of mitochondria relies on a sizable number of proteins. Many of these proteins are organized into complexes, which are located in the mitochondrial inner membrane. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a method for the isolation of intact protein complexes. Although it was initially used to study mitochondrial respiratory chain enzymes, it can also be applied to other protein complexes. The use of BN-PAGE has increased exponentially over the past few years and new applications have been developed. Here we review how to set up the basic system and outline modifications that can be applied to address specific research questions. Increasing the upper mass limit of complexes that can be separated by BN-PAGE can be achieved by using agarose instead of acrylamide. BN-PAGE can also be used to study assembly of mitochondrial protein complexes. Other applications include in-gel measurements of enzyme activity by histochemical staining and preparative native electrophoresis to isolate a protein complex. Finally, new ways of identifying protein spots in Blue Native gels using mass spectrometry are briefly discussed.     

Rhizobial nod gene-inducing activity in pea nodulation mutants: dissociation of nodulation and flavonoid response

With the characterization of the total genomes of Arabidopsis thaliana and Oryza sativa , several putative plasma membrane components have been identified. However, a lack of knowledge at the protein level, especially for hydrophobic proteins, have hampered analyses of physiological changes. To address whether protein complexes may be present in the native membrane, we subjected plasma membranes isolated from Spinacia oleracea leaves to blue-native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is well established in the separation of functional membrane protein complexes from mitochondria and chloroplasts, but a resolved protein complex pattern from PM of eukaryotic cells has previously not been reported. Using this method, protein complexes from Spinacia oleracea PM could be efficiently solubilized and separated, including the highly hydrophobic aquaporin (apparent molecular mass 230 kDa), a putative tetramer of H⁺-ATPase, and several less abundant complexes with apparent masses around or above 750 kDa. After denaturation and separation of the complexes into their subunits in a second dimension (SDS-PAGE), several of the complexes were identified as hydrophobic membrane proteins. Large amounts of protein (up to 1 mg) can be resolved in each lane, which suggests that the method could be used to study also low-abundance protein complexes, e.g. under different physiological conditions.     

Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach

Identification and characterization of multi-protein complexes is an important step toward an integrative view of protein-protein interaction networks that determine protein function and cell behavior. The limiting factor for identifying protein complexes is the method for their separation. Blue native PAGE (BN-PAGE) permits a high-resolution separation of multi-protein complexes under native conditions. To date, BN-PAGE has only been applicable to purified material. Here, we show that dialysis permits the analysis of multi-protein complexes of whole cellular lysates by BN-PAGE. We visualized different multi-protein complexes by immunoblotting including forms of the eukaryotic proteasome. Complex dynamics after gamma interferon stimulation of cells was studied, and an antibody shift assay was used to detect protein-protein interactions in BN-PAGE. Furthermore, we identified defined protein complexes of various proteins including the tumor suppressor p53 and c-Myc. Finally, we identified multi-protein complexes via mass spectrometry, showing that the method has a wide potential for functional proteomics.     

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.     

Gas chromatographic determination and mechanism of formation of D-amino acids occurring in fermented and roasted cocoa beans, cocoa powder, chocolate and cocoa shell

Summary. Pseudomonas sp. strain phDV1, being a phenol degrading bacterium, has been found to utilize phenol as sole carbon source via the meta pathway. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes involved in the aromatic degradation pathway. In particular, the Pseudomonas sp. strain phDV1 proteome was monitored under two different growth substrate conditions, using glucose or phenol as sole carbon source. The protein complexes map was compared by BN-PAGE after fractionation by sucrose density centrifugation of the cell extracts. Multiple differences were detected. Further, analysis and identification of the subunit composition of these complexes was carried out using MALDI-TOF MS, allowing the identification of 49 proteins. Additionally, functional information regarding protein–protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. Application of this functional proteomics method resulted in an higher number of the identified proteins.     

Mapping the proteome of thylakoid membranes by de novo sequencing of intermembrane peptide domains

The proteome of a membrane compartment has been investigated by de novo sequence analysis after tryptic in gel digestion. Protein complexes and corresponding protein subunits were separated by a 2-D Blue Native (BN)/SDS-PAGE system. The transmembrane proteins of thylakoid membranes from a higher plant (Hordeum vulgare L.) were identified by the primary sequence of hydrophilic intermembrane peptide domains using nano ESI-MS/MS-analysis. Peptide analysis revealed that lysine residues of membrane proteins are primarily situated in the intermembrane domains. We concluded that esterification of lysine residues with fluorescent dyes may open the opportunity to label membrane proteins still localized in native protein complexes within the membrane phase. We demonstrate that covalent labelling of membrane proteins with the fluorescent dye Cy3 allows high sensitive visualization of protein complexes after 2-D BN/SDS-PAGE. We show that pre-electrophoretic labelling of protein subunits supplements detection of proteins by post-electrophoretic staining with silver and CBB and assists in completing the identification of the membrane proteome.     

Blue native PAGE

Blue native PAGE (BN-PAGE) can be used for one-step isolation of protein complexes from biological membranes and total cell and tissue homogenates. It can also be used to determine native protein masses and oligomeric states and to identify physiological protein-protein interactions. Native complexes are recovered from gels by electroelution or diffusion and are used for 2D crystallization and electron microscopy or analyzed by in-gel activity assays or by native electroblotting and immunodetection. In this protocol, we describe methodology to perform BN-PAGE followed by (i) native extraction or native electroblotting of separated proteins, or (ii) a second dimension of tricine-SDS-PAGE or modified BN-PAGE, or (iii) a second dimension of isoelectric focusing (IEF) followed by a third dimension of tricine-SDS-PAGE for the separation of subunits of complexes. These protocols for 2D and 3D PAGE can be completed in 2 and 3 days.     

Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.     

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.     

The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein

Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H₂O₂). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin^−/− mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.     

Claudin-2 forms homodimers and is a component of a high molecular weight protein complex

Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.     

Identification of novel mitochondrial protein components of Chlamydomonas reinhardtii. A proteomic approach

Pure mitochondria of the photosynthetic alga Chlamydomonas reinhardtii were analyzed using blue native-polyacrylamide gel electrophoresis (BN-PAGE). The major oxidative phosphorylation complexes were resolved: F(1)F(0)-ATP synthase, NADH-ubiquinone oxidoreductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase. The oligomeric states of these complexes were determined. The F(1)F(0)-ATP synthase runs exclusively as a dimer, in contrast to the C. reinhardtii chloroplast enzyme, which is present as a monomer and subcomplexes. The sequence of a 60-kD protein, associated with the mitochondrial ATP synthase and with no known counterpart in any other organism, is reported. This protein may be related to the strong dimeric character of the algal F(1)F(0)-ATP synthase. The oxidative phosphorylation complexes resolved by BN-PAGE were separated into their subunits by second dimension sodium dodecyl sulfate-PAGE. A number of polypeptides were identified mainly on the basis of their N-terminal sequence. Core I and II subunits of complex III were characterized, and their proteolytic activities were predicted. Also, the heterodimeric nature of COXIIA and COXIIB subunits in cytochrome c oxidase was demonstrated. Other mitochondrial proteins like the chaperone HSP60, the alternative oxidase, the aconitase, and the ADP/ATP carrier were identified. BN-PAGE was also used to approach the analysis of the major chloroplast protein complexes of C. reinhardtii.     

An alternative strategy for the membrane proteome analysis of the green sulfur bacterium Chlorobium tepidum using blue native PAGE and 2-D PAGE on purified membranes

To avoid the specific problems concerning intrinsic membrane proteins in proteome analysis, an alternative strategy is described that is complementary to previous investigations using 2-D polyacrylamide gel electrophoresis (PAGE) techniques. The strategy involves (a) obtaining purified preparations of the membranes from Chlorobium tepidum by washing with 2 M NaBr, which removed membrane-associated soluble proteins and membrane-associated organelles; (b) separation of membrane protein complexes using 1-D Blue-native polyacrylamide gel electrophoresis (BN-PAGE) after solubilization with n-dodecyl-beta-d-maltoside (DDM); (c) combination of the BN with Tricine-SDS-PAGE; (d) high-throughput mass spectrometric analysis after gel band excision, in-gel digestion, and MALDI target spotting; and (e) protein identification from mixtures of tryptic peptides by peptide mass fingerprinting. Using this approach, we identified 143 different proteins, 70 of which have not been previously reported using 2-D PAGE techniques. Membrane proteins with up to 14 transmembrane helices were found, and this procedure proved to be efficient with proteins within a wide pI range (4.4-11.6). About 54% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, and protein translocation, while for the others, a function is not yet known, indicating the potential of the method for the elucidation of the membrane proteomes in general.     

Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes

In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS-PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS-PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS-PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS-PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS-PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.     

Intrinsic protein kinase activity in mitochondrial oxidative phosphorylation complexes

Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, the identity of the protein kinases mediating these phosphorylation events remains largely unknown. The detection of mitochondrial protein kinases is complicated by the low concentration of kinase relative to that of the target protein, the lack of specific antibodies, and contamination from associated, but nonmatrix, proteins. In this study, we use blue native gel electrophoresis (BN-PAGE) to isolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detect kinase activity, one-dimensional BN-PAGE gels were exposed to [γ-(32)P]ATP and then followed by sodium dodecyl sulfate gel electrophoresis. Dozens of mitochondrial proteins were labeled with (32)P in this setting, including all five complexes of oxidative phosphorylation and several citric acid cycle enzymes. The nearly ubiquitous (32)P protein labeling demonstrates protein kinase activity within each mitochondrial protein complex. The validity of this two-dimensional BN-PAGE method was demonstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and mass spectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases, suggesting a major role for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that these proteins undergo autophosphorylation and, to a lesser degree, tenacious (32)P-metabolite association. In-gel Complex IV activity was shown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for protein phosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain an autophosphorylation mechanism, which may play a functional role in the regulation of these multiprotein units.