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.     

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.     

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.     

Low-SDS Blue native PAGE

SDS normally is strictly avoided during Blue native (BN) PAGE because it leads to disassembly of protein complexes and unfolding of proteins. Here, we report a modified BN-PAGE procedure, which is based on low-SDS treatment of biological samples prior to native gel electrophoresis. Using mitochondrial OXPHOS complexes from Arabidopsis as a model system, low SDS concentrations are shown to partially dissect protein complexes in a very defined and reproducible way. If combined with 2-D BN/SDS-PAGE, generated subcomplexes and their subunits can be systematically investigated, allowing insights into the internal architecture of protein complexes. Furthermore, a 3-D BN/low-SDS BN/SDS-PAGE system is introduced to facilitate structural analysis of individual protein complexes without their previous purification.     

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.     

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.     

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.     

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.     

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.     

Analysis of membrane protein complexes by blue native PAGE

Blue native polyacryamide gel electrophoresis is a special case of native electrophoresis for high resolution separation of enzymatically active protein complexes from tissue homogenates and cell fractions. The method is powerful between 10 and 10,000 kDa. Also membrane protein complexes are separated well after solubilization of complexes with mild neutral detergents. The separation principle relies on binding of Coomassie blue G250 which provides negative charges to the surface of the protein. During migration to the anode, protein complexes are separated according to molecular mass and/or size and high resolution is obtained by the decreasing pore size of a polyacrylamide gradient gel. The principles of 2-dimensional blue native sodium dodecyl sulfate polyacrylamide gel electrophoresis are presented here together with a practical step-by-step guide to performing the method in the laboratory.     

Proteomic approach to characterize the supramolecular organization of photosystems in higher plants

A project to investigate the supramolecular structure of photosystems was initiated, which is based on protein solubilizations by digitonin, protein separations by Blue native (BN)-polyacrylamide gel electrophoresis (PAGE) and protein identifications by mass spectrometry (MS). Under the conditions applied, nine photosystem supercomplexes could be described for chloroplasts of Arabidopsis, which have apparent molecular masses between 600 and 3200 kDa on BN gels. Identities of the supercomplexes were determined on the basis of their subunit compositions as documented by 2D BN/SDS-PAGE and BN/BN-PAGE. Two supercomplexes of 1060 and approximately 1600 kDa represent dimeric and trimeric forms of photosystem I (PSI), which include tightly bound LHCI proteins. Compared to monomeric PSI, these protein complexes are of low abundance. In contrast, photosystem II mainly forms part of dominant supercomplexes of 850, 1000, 1050 and 1300 kDa. According to our interpretation, these supercomplexes contain dimeric PSII, 1-4 LHCII trimers and additionally monomeric LHCII proteins. The 1300-kDa PSII supercomplex (containing four LHCII trimers) is partially converted into the 1000-kDa PSII supercomplex (containing two LHCII trimers) in the presence of dodecylmaltoside on 2D BN/BN gels. Analyses of peptides of the trypsinated 1300-kDa PSII supercomplex by mass spectrometry allowed to identify known subunits of the PSII core complex and additionally LHCII proteins encoded by eight different genes in Arabidopsis. Further application of this experimental approach will allow new insights into the supermolecular organization of photosystems in plants.     

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.     

Blue native electrophoresis as a tool for detection of PIP-containing protein complexes in the plasma membranes

Blue native electrophoresis (BN-PAGE) is presently considered as one of effective methods for the identification of membrane protein complexes. The choice of a nonionic detergent and the detergent to protein ratio are critically important. Our experiments with plasma membranes of etiolated pea (Pisum sativum L.) seedlings showed that various nonionic detergents—digitonin, dodecyl maltoside, and Triton X-100—solubilized similar assortments of protein complexes. Irrespective of the detergent type, PIP aquaporins were always observed in the 440-kD protein complex. Only in the case of dodecyl maltoside, the PIP aquaporins were also revealed in the complexes with the lower and higher molecular weights when the detergent/protein ratio increased.     

Application of "blue native" electrophoresis in the studies of mitochondrial respiratory chain complexes in physiology and pathology

"Blue Native" polyacrylamide gel electrophoresis (BN-PAGE), originally described by Sch?gger and von Jagow in 1991, is an elegant method to study protein complexes from mitochondrial membranes. BN-PAGE, commonly used in molecular biology to study composition of protein complexes and protein-protein interactions, enables separation of respiratory chain complexes keeping their properties and enzymatic activities unchanged. BN-PAGE, supplemented by other methods, e.g. in gel activity assay, SDS-PAGE (as a first or second dimension) can be successfully adapted for diagnosis of mitochondrial diseases connected with abnormalities of the respiratory chain. Therefore, to make a correct diagnosis of the deficiency of respiratory chain complexes, other methods, as histochemical colorimetric reactions allowing evaluation of the OXPHOS catalytic activity in individual cells and spectrophotometric technique should be used simultaneously with BN-PAGE.     

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.     

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.     

Localization of 13 one-helix integral membrane proteins in photosystem II subcomplexes

Photosystem II is a multimeric protein complex of the thylakoid membrane in chloroplasts. Approximately half of the at least 26 different integral membrane protein subunits have molecular masses lower than 10 kDa. After one-dimensional (1D) or two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) separation, followed by enzymatic digestion of detected proteins, hardly any of these low-molecular-weight (LMW) subunits are detectable. Therefore, we developed a method for the analysis of highly hydrophobic LMW proteins. Intact proteins are extracted from acrylamide gels using a mixture of formic acid and organic solvent, precipitated with acetone, and analyzed by “top-down” mass spectrometry (MS). After offline nanoESI (electrospray ionization) MS, all LMW one-helix proteins from photosystem II were detected. In the four detected photosystem II supercomplexes of Nicotiana tabacum wild-type plants, 11 different one-helix proteins were identified as PsbE, -F, -H, -I, -K, -L, -M, -Tc, -W, and two isoforms of PsbX. The proteins PsbJ, -Y1, and -Y2 were localized in the buffer front after blue native (BN) PAGE, indicating their release during solubilization. Assembled PsbW is detected exclusively in supercomplexes, whereas it is absent in photosystem II core complexes, corroborating the protein’s function for assembly of the light-harvesting complexes. This approach will substantiate gel-blot immunoanalysis for localization and identification of LMW protein subunits in any membrane protein complex.     

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.     

Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling

Blue native (BN) gel electrophoresis is a powerful method for protein separation. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), it enables large scale identification of protein complexes and their subunits. Current BN-MS approaches, however, are limited in size resolution, comprehensiveness, and quantification. Here, we present a new methodology combining defined sub-millimeter slicing of BN gels by a cryo-microtome with high performance LC-MS/MS and label-free quantification of protein amounts. Application of this cryo-slicing BN-MS approach to mitochondria from rat brain demonstrated a high degree of comprehensiveness, accuracy, and size resolution. The technique provided abundance-mass profiles for 774 mitochondrial proteins, including all canonical subunits of the oxidative respiratory chain assembled into 13 distinct (super-)complexes. Moreover, the data revealed COX7R as a constitutive subunit of distinct super-complexes and identified novel assemblies of voltage-dependent anion channels/porins and TOM proteins. Together, cryo-slicing BN-MS enables quantitative profiling of complexomes with resolution close to the limits of native gel electrophoresis.Blue native (BN)¹-PAGE and its colorless variant, colorless native PAGE, were originally developed by Schägger and co-workers as end point separation methods for characterization of solubilized mitochondrial membrane protein (super-)complexes under close-to-native conditions (1–3). Subsequently, native gel electrophoresis became the method of choice for first dimension separation followed by second dimension SDS-PAGE in two-dimensional gel-based proteomic analyses (2D-BN) of membrane protein complexes. After staining of the gel-separated proteins, protein spots are individually analyzed by different mass spectrometric methods, and the identified proteins were assigned to complexes based on their co-migration pattern (2D-BN-MS (4)). However, these 2D-BN-MS approaches exhibit the following severe shortcomings: (i) they are critically dependent on the staining properties of individual proteins; (ii) the size resolution of protein complexes is low; and (iii) the assignment of identified proteins to spots and complexes may be ambiguous. Therefore, application of 2D-BN-MS has remained largely restricted to the characterization of highly abundant and well defined membrane protein complexes such as complexes I–V of the respiratory chain in mitochondria (5–7), photosynthetic complexes (8–10), or viruses (11).In a first attempt to overcome these shortcomings of 2D-BN-MS, Wessels et al. (12) coupled BN-PAGE separation more directly to MS analysis by manually cutting the gel lane into 24 slices/sections of about 2 mm width that were separately digested and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Their study on HEK cell mitochondria identified 59 of the 90 canonical subunits of the oxidative respiratory chain (OXPHOS) complexes I–V. The respective protein abundance profiles (based on standard label-free quantification) showed clustering of their peak maxima into the expected complexes I–V. Since then, this one-dimensional BN-MS methodology has been gradually improved with respect to quality of the native gel separation, LC-MS/MS sensitivity, and robustness of the quantitative evaluation. Thus, two recent studies on human mitochondrial preparations (each analyzing two BN separations in 60 and 24 slices, respectively) reported identification and hierarchical profile clustering of 464 (13) and 437 (14) mitochondrial proteins. In these studies, 82/73 (including 8 single-peptide hits) and 55/54 (including 7 single-peptide hits) of the 90 known OXPHOS complex subunits were identified/clustered, respectively. Furthermore, TMEM126B was identified as a novel and essential subunit of an OXPHOS complex I assembly complex (13).Notably, all of these studies achieved clustering of protein profiles for the dominating populations of complexes, although they largely failed to obtain information on sub-complexes and super-complexes, most likely as a consequence of the strong undersampling in the first dimension (well below the resolution of BN-PAGE) and a limited dynamic range of MS-based identification and quantification.To improve the resolution of BN-MS for analysis of protein super-complexes and their subunit composition, we have recently started to develop sub-millimeter sampling of BN gel lane sections by using cryo-microtome slicing (15, 16). Here, we describe a new methodology for comprehensive and high resolution complexome profiling that combines this high resolution gel sampling method with a sensitive and precise label-free MS quantification workflow. Protein profiles determined in a mammalian mitochondrial membrane preparation showed a highly effective mass resolution (<5% molecular weight difference) over the whole BN-PAGE separation range and together covered a major portion of the mitochondrial membrane proteome.