This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.
The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.
Mitochondrial energy in the form of ATP is not only essential for cell survival, but also for the regulation of cell death. The generation of ATP by oxidative phosphorylation requires a functional electron transport chain (ETC; Cx-I to IV) and mitochondrial ATP synthase (Cx-V). Recent studies have shown that these large protein complexes are organized into supercomplexes, called respirasomes and synthasomes1,2. It is challenging to analyze the assembly, dynamics, and activity regulation of these massive complexes and supercomplexes. While oxygen consumption measurements taken with an oxygen electrode and enzyme assays conducted using a spectrophotometer can give valuable information about ETC complex activity, these assays cannot provide information regarding the presence, size, and subunit composition of the protein complex or supercomplexes involved. However, the development of blue and clear native (BN and CN, respectively) PAGE3 has created a powerful tool for revealing important information about complex composition and assembly/disassembly and about the dynamic regulation of the supramolecular organization of these vital respiratory complexes under physiological and pathological conditions4.
The assembly of these complexes into higher-order supercomplexes appears to regulate mitochondrial structure and function5. For example, respirasome assembly increases the efficiency of electron transfer and the generation of the proton motive force across the mitochondrial inner membrane5. In addition, the assembly of synthasomes not only increases the efficiency of ATP production and the transfer of energy equivalents into the cytoplasm2, but it also molds the mitochondrial inner membrane into the tubular cristae6,7. Studies of supercomplex assembly during cardiac development in mouse embryos show that the generation of Cx-I-containing supercomplexes in the heart begins at about embryonic day 13.58. Others have shown that the amount of Cx-I-containing supercomplexes decreases in the heart due to aging or ischemia/reperfusion injuries9,10 or may play a role in the progression of neurodegenerative diseases11.
This protocol describes methods for native gel PAGE that can be used to investigate the assembly and activity of the ETC complexes and supercomplexes. The approximate molecular weight of mitochondrial supercomplexes can be assessed by separating the protein complexes in CN or BN polyacrylamide gels. CN PAGE also allows for the visualization of the enzymatic activity of all mitochondrial complexes directly in the gel (in-gel assays; IGA)12. This work demonstrates the activity of respirasomes by highlighting the ability of Cx-I to oxidize NADH through IGA and the presence of synthasomes due to the ATP-hydrolyzing activity of Cx-V by IGA. The multiple complexes and supercomplexes containing Cx-I and Cx-V can also be demonstrated by transferring the proteins onto nitrocellulose membranes and performing immunoblotting. The advantage of this approach is that BN or CN PAGE generally separates protein complexes based on their physiological size and composition; the transfer to a membrane preserves this pattern of bands. Analyzing protein complexes in a BN or CN PAGE can also be done using 2D-PAGE (see Fiala et al.13 for a demonstration) or by sucrose density centrifugation14,15. To further analyze a specific band, it can be excised from the BN PAGE, and the proteins from this protein complex can be purified by electroeluting them under native conditions. Native electroelution can be performed within a few hours, which could make a significant difference to the passive diffusion (as used in Reference 16) of proteins from a gel into the surrounding buffer.
In summary, these methods describe several approaches that allow for the further characterization of high-molecular-weight supercomplexes from mitochondrial membranes.
All experiments were performed using hearts from C57BL/6N mice (wild type). Mice were anesthetized with CO2 prior to cervical dislocation, and all procedures were performed in strict accordance with the Division of Laboratory Animal Medicine at the University of Rochester and in compliance with state law, federal statute, and NIH policy. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Rochester (University Committee on Animal Resources).
1. CN and BN PAGE
NOTE: All equipment used for BN and CN PAGE must be free of detergent. To ensure this, wash all equipment with 0.1 M hydrochloric acid, followed by extensively rinsing with deionized H2O.
3 % to 8 % (mini) | 4 % to 10 % (maxi) | |||
0.5 gels (light) | 0.5 gels (heavy) | 0.5 gels (light) | 0.5 gels (heavy) | |
AAB (mL) | 0.42 | 1.3 | 2.5 | 7.7 |
CN/BN buffer (mL) | 1.6 | 1.6 | 8.5 | 8.5 |
H2O (mL) | 2.7 | 1.4 | 14 | 6.3 |
Glycerol (g) | 0 | 0.47 | 0 | 2.5 |
Volume (mL) | 4.72 | 4.77 | 25 | 25 |
APS (µL) | 27 | 27 | 65 | 65 |
TEMED (µL) | 4 | 4 | 10 | 10 |
Table 1: Quantities of Ingredients Needed to Pour 1 Mini- or Maxi-PAGE. The volumes used in this table are calculated for 1 mini- or 1 maxi-gel, 1.5 mm thick. The volume of AAB is based on a 40% stock solution. Light and heavy refer to the concentration of AAB. APS and TEMED are added after each column of the gradient mixer is filled with AAB solution.
2. In-gel Assays for Cx-I and Cx-V
NOTE: The assays are performed at room temperature. Take photos, scans, or images of the developing bands for documentation. (Important) Proteins cannot not be transferred onto nitrocellulose membranes after completing an IGA.
3. Protein Transfer to Nitrocellulose or Polyvinylidene Difluoride (PVDF) Membranes
4. Immunoblotting
5. Electroelution
To visualize mitochondrial supercomplexes, freshly isolated mitochondria from mice were used17,18. Mitochondrial supercomplexes are sensitive to repeated cycles of freezing and thawing, leading to their disintegration, although this may be tolerable for some researchers. If freezing is necessary for storage, to ensure best results, samples should not undergo more than one cycle of freezing and thawing.
To visualize the mitochondrial ETC complexes with BN PAGE, 100 µg of protein from isolated heart mitochondria were loaded onto a 4-10% gel (Figure 1A). The Coomassie stain in the loading and cathode buffer is sufficient to label the protein complexes during the run. Supercomplexes appear after increasing the contrast digitally (not shown). For CN PAGE, two samples of 20 µg of protein from isolated mitochondria were loaded onto a 3 – 8% CN gel and separated (Figure 1B). The CN PAGE was stained with Coomassie and destained to visualize the protein complexes. After increasing the contrast digitally, several protein complexes with a molecular weight greater than the monomer of Cx-I appeared (Figure 1B, right). The concentrations of AAB used allowed for the largest supercomplexes to just enter the gel from the well. However, a gel ending with a gradient of less than 3% AAB is not stable enough to manipulate for transfer or for excising a band or lane. In addition, the low concentration of AAB in the upper parts of the 3-8% CN gels maintains some mobility of the protein complexes, which is important if considering native electroelution19.
Monomers and supercomplexes of Cx-I and monomers, dimers, and supercomplexes of Cx-V are enzymatically active and can be visualized by IGA (Figure 1C-F). The assays show that, in isolated heart mitochondria, Cx-I and Cx-V are present in protein complexes greater than their respective monomers. In the IGA assay for Cx-I, NADH is oxidized and electrons are transferred to reduce nitroblue tetrazolium. This results in a localized blue color at the molecular weight of the Cx-I monomers and the Cx-I-containing respirasomes/supercomplexes (Figure 1C). The activity of Cx-V is assessed from the ability of the F1 subunit to hydrolyze ATP and can be done using CN or BN gels (Figure 1D-F). The ADP generated from this reaction interacts with lead and results in a white precipitate at the level of Cx-V monomers, dimers, synthasomes, and subcomplexes (most likely the unassembled F1 portion of Cx-V). Note that oligomycin eliminates the labeling of these bands, confirming that they contain Cx-V (Figure 1E).
For all experiments described here, the zwitterionic detergent, lauryl maltoside, was used at a concentration of 2 µg/1 µg of protein, which is the highest possible concentration that preserves the supercomplexes while providing consistent and reproducible results (Figure 1F). However, the effectiveness of lauryl maltoside depends on the lot number, storage conditions, and age. Thus, the exact concentration used in one laboratory is not necessarily the same as reported in manuscripts. The proper concentration of detergent will solubilize the membranes but keep the complexes and supercomplexes intact and must be determined by using a variety of concentrations of lauryl maltoside (Figure 1F). For CN PAGE, a total sample volume of 40 µL per well was prepared here, resulting in a protein/detergent ratio of 1 µg/2 µg or a detergent/buffer ratio of 1 µg/1 µL (equal to 1.9 mM). From the 40 µL, 30 µL was applied per well of the mini-gel; the remaining was used as an aliquot for the detection of VDAC, a loading control (Figure 2D).
For immunoblotting, CN is preferred to BN PAGE because the proteins are not loaded with Coomassie, which may interfere with detection by antibodies. Figure 2 shows the detection of supercomplexes containing the Cx-I and Cx-V proteins NDUFB6 and ATP5A from isolated heart, liver, and brain mitochondria. Ponceau S labeling after transfer and before immunoblotting can be used to mark the molecular weight markers and to control for protein loading (Figure 2A). This will visualize the monomers of the protein complexes of the ETC on nitrocellulose membranes, but Ponceau S labeling is not always sufficient for the visualization of mitochondrial supercomplexes (Figure 2A), which can be achieved by immunoblotting.
Cx-I does not assemble into dimers and tetramers, per se, but forms increasingly higher molecular-weight supercomplexes with Cx-III and Cx-IV14,20,21. Here, using an antibody against NDUFB6 shows that the sample from the heart contained more Cx-I monomer and high-molecular-weight supercomplexes (top bands) that the mitochondria from the liver or the brain (Figure 2B). The amount of mid-range respirasome supercomplexes was also much higher in the heart than in the other tissues (Figure 2B).
Using anti-ATP5A antibodies, monomers of Cx-V are detectable in mitochondria from all tissues, while a distinct pattern of bands representative of dimers (D) and larger supercomplexes (SC), which are clearly visible in heart mitochondria, are not as prominent in liver and brain mitochondria (Figure 2C). Overexposure (1 min versus 20 s) of the immunoblot shows several Cx-V-containing supercomplexes, which could represent tetramers and synthasomes (Figure 2C). These patterns of Cx-V-containing protein complexes in heart, liver, and brain mitochondria show differences that may be tissue-specific and have not yet been explored.
Protein loading of these blots can be followed by Ponceau S staining and VDAC detection of the abovementioned aliquot by SDS PAGE, as demonstrated in Figure 2D.
Not all antibodies are suitable to detect a protein within the quaternary or tertiary structure of a protein complex after native PAGE. To overcome this problem, entire and partial lanes from the native gel can be mounted on a denaturing gel for a second dimension (2D gels, see Reference 13 for a demonstration). 2D electrophoresis is a valuable tool for visualizing proteins in a supramolecular complex. However, as shown in Figure 2, supercomplexes are present in variable amounts, and the signal of individual proteins from supercomplexes may be hard to visualize in the second, denaturing dimension. To overcome this problem, the electroelution of protein complexes from native gels was used here. This isolates supercomplex bands from multiple lanes to yield more material for further study.
When using electroelution, only the band of interest, which has been identified by IGA and/or visualized on a BN PAGE, is excised; the proteins from this piece of gel are further purified by elution from the gel. Figure 3A shows a lane of a BN PAGE from which bands representing the monomer were excised for electroelution. To assess the Cx-V activity of the monomer after electroelution, the eluate was applied to a second CN PAGE. The eluate of the monomer still contains enzymatically active Cx-V, but subcomplexes also appear (Figure 3B). Silver staining of the eluate after native CN PAGE (Figure 3C) or denaturing SDS PAGE (Figure 3D) indicates the presence of proteins in the eluate, and immunoblotting against ATP5A indicates the presence of Cx-V in both samples (Figure 3D).
Figure 1: Visualization of Mitochondrial Supercomplexes. (A) Two lanes of a 4-10% BN maxi-gel, with samples of isolated heart mitochondria. Aliquots of the same sample were run in both lanes, at 100 µg of protein per well. The monomers of protein complexes I, II, III, IV, and V of the ETC are clearly visible and are labeled to the right of the gel. (B) Two samples of heart mitochondria (1 and 2, 20 µg of protein/lane) were separated on a CN PAGE and visualized by Coomassie staining. SC indicates the position of the supercomplexes after magnification and digital enhancement of the upper part of the gel (the area is indicated by red arrows). (C) 20 µg of mitochondrial protein were separated on a 3-8% CN PAGE and were processed for Cx-I IGA. Magnified and digitally enhanced images demonstrate bands of Cx-I reaction product. (D) 20 µg of mitochondrial protein were separated on a 3-8% BN PAGE and processed for Cx-V IGA. Magnified and digitally enhanced images demonstrate bands of Cx-V reaction product. (E) 50 µg of mitochondrial protein were separated on a 5-15% CN PAGE and processed for Cx-V IGA without (-) and with (+) 5 µg/mL oligomycin (Oligo). (F) Mitochondrial protein (20 µg/lane) was solubilized with 2 – 6 µg of lauryl maltoside/1 µg of protein, as indicated at the top of the gel, and separated on a 3-8% CN gel followed by Cx-V IGA. All images were photographed using either a light table (A–C) or a black surface (D–F). Camera specifications are in the Table of Materials. The location of the molecular weight markers (MW) are indicated on the left side of all panels. Abbreviations: D = dimers; F1* = subcomplex of Cx-V; LM = lauryl maltoside; m = molecular weight marker; M = monomers; SC = supercomplexes. Please click here to view a larger version of this figure.
Figure 2: Detection of Mitochondrial Supercomplexes by Immunoblotting. 20 µg of protein from the heart (H), liver (Li), and brain (B) mitochondria were separated on a 3-8% CN PAGE and transferred onto nitrocellulose membrane. (A) Ponceau S staining of the membrane indicates the presence of protein complexes and molecular weight markers (m). The blue arrow points to the top of the gel. (B) The Cx-I protein, NDUFB6, was immunolabeled on the blot shown in (A) (1 min exposure time). (C) Cx-V was visualized with anti-ATP5A antibody (20 s and 1-min exposure, as indicated). The red arrows in (B) and (C) indicate the area magnified and digitally enhanced for the visualization of Cx-I- and Cx-V-containing supercomplexes, and the blue arrow points to the top of the gel. (D) Ponceau S and immunolabeling of the VDAC aliquot of each extract used in (A), (B), and (C), which were separated by SDS PAGE and transferred to nitrocellulose. Abbreviations: M = monomers of Cx-I or Cx-V, D = dimers of Cx-V, SC = supercomplexes containing Cx-I or Cx-V. Please click here to view a larger version of this figure.
Figure 3: Electroelution of Cx-V. (A) BN PAGE of a mitochondrial sample. The boxed band represents the monomer of Cx-V that was excised and electroeluted. (B) The eluate was subjected to CN PAGE, and subsequent IGA for Cx-V demonstrates monomers (M) and subcomplexes containing F1 of Cx-V. (C) Silver staining of the CN PAGE of the eluate demonstrates monomers (M). (D) Silver staining (left panel) and immunoblot for ATP5A (right panel) of a denaturing sodium dodecyl sulfate (SDS) PAGE of the eluate indicates the presence of Cx-V. For SDS PAGE, please refer to protocols published elsewhere. Please click here to view a larger version of this figure.
A functional ETC is necessary for mitochondrial ATP generation. The complexes of the ETC are able to form two types of supercomplexes: the respirasomes (Cx-I, -III, and -IV)1 and the synthasomes (Cx-V)2. The assembly of each complex is required for an intact ETC, while the organization of the ETC into supercomplexes is thought to increase overall ETC efficiency5,22. How these supercomplexes assemble and disassemble is not well understood, but the protocols presented here may allow for a better understanding of these processes.
The major challenge of studying ETC assembly is the size of these protein complexes. For example, mitochondrial respirasomes may consist of Cx-I (about 880 kDa) and one or more molecules of Cx-III (460 kDa) and Cx-IV (200 kDa, active as a dimer), resulting in a supercomplex with a molecular weight of about 2,000 kDa. In addition, Cx-V has a molecular weight of about 600 kDa but has been shown to assemble into dimers, tetramers, oligomers, and ribbons of dimers7,23, resulting in supercomplexes with a molecular weight of at least 2,000 kDa. Considering the enormous size of these supercomplexes, several partly related approaches have traditionally been used to identify and to characterize these supercomplexes, by this lab and by others8,14,24.
This work has demonstrated the technique of native gel PAGE, where active protein complexes are gently extracted from the mitochondrial membrane using mild detergents. The complexes migrate in the gels primarily due to their size and intrinsic charge (CN PAGE) or due to the size and negative charge of protein-bound Coomassie blue (BN PAGE). These gels can be stained using Coomassie blue (e.g., in CN gels) or silver stain (e.g., in BN and CN PAGE) to reveal proteins bands.
Lauryl maltoside was used for the experiments demonstrated here because this detergent worked most reliably. Alternatively, digitonin can be used to extract protein complexes12. Data from mitochondria isolated from adult hearts, livers, and brains have been shown here, but these techniques have been performed on embryonic and adult heart homogenates8. Others have performed this technique using isolated mitochondria from cultured cells24. However, when using tissue homogenates or cultured cells with a high DNA content, it may be useful to add a nuclease to prevent streaking during electrophoresis25.
The activity of the different complexes can be assayed directly in the gel, as demonstrated here for Cx-I and Cx-V, and techniques are also available to examine the activities of Cx-II, -III, and -IV12. However, analyzing these IGAs must be done carefully. First, the enzymatic reaction could be due to non-ETC enzymes or incompletely assembled complexes. For example, it is routine to perform the Cx-V IGA with a parallel gel that has been treated with oligomycin to inhibit intact Cx-V (Figure 3). In addition, other NADH oxidases may account for Cx-1 in-gel activity. One might perform a parallel IGA in the presence of a Cx-I inhibitor, such as rotenone. However, in Figure 1, isolated mitochondria were used, so cytoplasmic NADH oxidases were not present; thus, the data most likely represents Cx-I-containing monomers and supercomplexes. In addition, the quantification of these results is possible by measuring the signal intensity of the bands on photographs or scans. Drawbacks to this approach include the differences between and within gels, the mobility of the reaction product, the inability to adequately quantify the product throughout the depth of the gel, and the potential non-linearity of the reaction27. To overcome the latter, some have suggested obtaining rates of reactions using serial photographs, but this has not been tried here.
The protein in native gels can also be transferred to membranes, and the protein composition of the bands can be examined by immunoblotting (demonstrated here) or 2D PAGE (demonstrated in reference13). Monomers of the ETC complexes have traditionally been identified by their abundance in native gels of isolated mitochondria, their location in the gel, and their position relative to the molecular marker protein (Figure 1). Furthermore, the labeling of subsequent immunoblots with antibodies specific for subunits of different complexes helps to identify the complex and the composition of supercomplexes. Therefore, anti-NDUFB6 and -ATP5A identify monomers of and supercomplexes containing Cx-1 and Cx-V, respectively, as demonstrated here. Antibodies to Cx-II to IV can be used for the same purpose. In some cases, antibodies that work well in denaturing gels may not work well in native gels due to the fact that some antibodies are specific to denatured protein or that an epitope can be masked by other proteins in a native complex.
The exact determination of the molecular weight of these supercomplexes is difficult, since most available molecular weight markers lie below the size of Cx-V monomers. The migration of protein complexes in a native gel depends on the size, intrinsic charge, and detergent used28. In the examples here, monomers of the complexes were identified based on gels, markers, and immunoblotting. Dimers of Cx-V were identified based on relative migration compared to monomers of Cx-I and -V and on immunoblotting. Everything else above the monomers and dimers in gels, IGAs, and immunoblots are considered to be supercomplexes.
Native immunoblots may also be quantified for supercomplexes by analyzing band density using standard techniques. This method was not shown here, but a recent publication demonstrates this technique8. Normalization of protein loading may be done using the Ponceau red staining of the lane or by saving a sample of the mitochondrial extract to measure the density of the VDAC band on a denaturing immunoblot (Figure 2A and D). Furthermore, examining the ratio of supercomplexes to monomers in the same immunoblot is another way to quantify band density, but one must be careful to take pictures at different times during blot development so that the bands are not over-exposed.
Finally, bands from native gels can be electroeluted to generate purified, active protein complexes that can assemble into higher-order complexes and can be used for further studies of complex function. For example, Cx-V monomers were previously electroeluted and reconstituted into liposomes to demonstrate the electrical functionality29. An alternative approach to native electroelution is elution by passive diffusion into the surrounding buffer16, but this is slow compared to native electroelution. Finally, a major problem with electroelution is maintaining the enzymatic function of the electroeluted protein complex, as the complex may dissociate during purification. Therefore, any assay of function after isolation by this technique would have to be rigorously tested.
By combining these protocols with enzymatic assays and oxygen consumption measurements, which probe the function of individual ETC complexes and the activity of the entire ETC30, and other methods, such as the crystallographic31 and electron microscopic32 evaluation of the structure of complexes and supercomplexes, a completer picture of the inner workings of the ETC can and has emerged. We are closer to understanding how the complexes are assembled, how electrons flow down the chain, and how protons are pumped across the membrane to generate the gradient and then flow back to the matrix through Cx-V to generate ATP. Undoubtedly, these techniques will be further refined to provide additional details about the structure, function, and dynamic regulation of the ETC.
The authors have nothing to disclose.
This work was supported by grants from the American Heart Association Founder’s Affiliate [12GRNT12060233] and the Strong Children’s Research Center at the University of Rochester.
Protean II mini-gel chamber | Biorad | 1658004 | Complete set to pour and run mini-gel electrophoresis |
Protean XL maxi-gel | Biorad | 1653189 | Complete set to pour and run maxi-gel electrophoresis |
Gradient maker, Hoefer SG15 | VWR | 95044-704 | Pouring mini-gel gradients |
Gradient maker, maxi-gel | VWR | GM-100 | Pouring maxi-gel gradients |
Transfer kit | Biorad | 1703930 | Complete set to wet transfer of proteins onto membranes |
Electroeluter model 422 | Biorad | 1652976 | Electroelution of proteins from native or SDS PAGES |
Glass plates | Biorad | 1653308 | Short plates |
Glass plates | Biorad | 1653312 | Spacer plates |
Glass plates | Biorad | 1651823 | Inner plates |
Glass plates | Biorad | 1651824 | Outer Plates |
Power supply | Biorad | 1645070 | Power supply suitable for native electrophoresis |
ECL-Western | Thermo Scientific | 32209 | Chemolumniscense substrate |
SuperSignal-West Dura | Thermo Scientific | 34075 | Enhanced chemolumniscense substrate |
Film/autoradiography film | GE Health care | 28906845 | Documentation of Western blots |
Film processor CP1000 | Agfa | NC0872640 | |
Canon Power Shot 640 | Canon | NA | Taking photos to document gels, membranes and blots. |
Canon Power Shot 640 Camera hood | Canon | shielding camera for photos being taken on a light table | |
Acrylamide/bisacrylamide | Biorad | 1610148 | 40% pre-mixed solution |
Glycine | Sigma | G7403 | |
SDS (sodium dodecyl sulfate) | Invitrogen | 15525-017 | |
Tris-base | Sigma | T1503 | Buffer |
Tricine | Sigma | T0377 | |
Sodium deoxychelate | Sigma | D66750 | Detergent |
EDTA | Sigma | E5134 | |
Sucrose | Sigma | S9378 | |
MOPS | Sigma | M1254 | Buffer |
Imidazole | Sigma | I15513 | Buffer |
Lauryl maltoside | Sigma | D4641 | Detergent |
Coomassie G250 | Biorad | 161-0406 | |
Aminohexanoic acid | Sigma | O7260 | |
Native molecular weight kit | GE Health care | 17-0445-01 | High molecular weight calibraition kit for native electrophoresis. |
Name | Company | Catalog Number | Comments |
NADH | Sigma | N4505 | |
Nitroblue tetrazolium | Sigma | N6639 | |
Tris HCL | Sigma | T3253 | |
ATP | Sigma | A2383 | |
Name | Company | Catalog Number | Comments |
Lead(II) nitrate (Pb(NO3)2): | Sigma | 228621 | |
Oligomycin | Sigma | O4876 | |
Name | Company | Catalog Number | Comments |
Ponceau S | Sigma | P3504 | |
anti-ATP5A | Abcam | ab14748 | antibody to ATP synthase subunit ATP5A |
anti-NDUFB6 | Abcam | ab110244 | antibody to Cx-1 subunit NDUFB6 |
anti-VDAC | Calbiochem | 529534 | antibody to VDAC |
ECL HRP linked antibody | GE Health Care | NA931V | secondary antibody to ATP5A, NDUFB6 and VDAC |
Blocking reagent | Biorad | 170-6404 | |
BSA | |||
sodium chloride | Sigma | S9888 | |
potassium chloride | Sigma | P9541 | |
EGTA | Sigma | E3889 | |
Name | Company | Catalog Number | Comments |
Silver staining Kit | Invitrogen | LC6070 |