A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.
Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.
BN-PAGE separation was first directly coupled to LC-MS analysis (BN-MS) by the Majeran1 and Wessels2 research groups using manual slicing of BN-PAGE gel lanes. Their analyses identified a number of abundant membrane protein complexes with known subunit composition from plant plastids and HEK cell mitochondria, respectively. However, these analyses were far from comprehensive and did not allow for unbiased identification of novel assemblies. Performance of mass spectrometers and label-free quantification methods has considerably improved since then, which has enabled comprehensive BN-MS analyses. This has coined the term "complexome profiling". For example, Heide and coworkers analyzed rat heart mitochondria identifying and clustering 464 mitochondrial proteins, thereby confirming many known assemblies. In addition, they found TMEM126B to be a novel and crucial subunit of a specific assembly complex3. Comparable results (with 437 mitochondrial protein profiles) were obtained in a parallel study of HEK cell mitochondria4.
Despite these improvements, several issues have remained that restrain the full potential of BN-MS for complexome profiling. A major limitation is the effective size resolution of complexes that is determined by two factors: the (i) quality of the BN-PAGE separation, which depends on the uniformity of the gel matrix pore gradient as well as the stability/solubility of the sample complexes, and (ii) step size of gel sampling, which is at best 1 mm when using conventional manual slicing5,6. Poor size resolution not only misses subtle complex isoforms and heterogeneities, but it also negatively impacts the dynamic range and confidence of unbiased, de novo subunit assignment and quantification.
Other challenges include the precision of protein quantification and coverage of the actual dynamic range of protein abundances in the sample by mass spectrometric analysis. Therefore, application of BN-MS complexome profiling has remained largely restricted to biological samples with lower complexity, high expression of target complexes, and favorable solubilization properties (i.e., plastids, mitochondria, and microorganisms)6,7,8,9,10.
We recently introduced cryomicrotome slicing-assisted BN-MS (csBN-MS), which combines precise sub-millimeter sampling of BN-PAGE gel lanes with comprehensive MS analysis and elaborate MS data processing for determination of protein profiles with high confidence11. Application to a mitochondrial membrane preparation from rat brains demonstrated previously unmet effective complex size resolution and maximum coverage of oxidative respiratory chain complex (OXPHOS) subunits (i.e., 90 of 90 MS-accessible). This example also identified a number of novel protein assemblies.
Described here are optimized procedures for preparative scale BN-PAGE separation of protein complexes (not restricted to a particular biological source), casting of large preparative BN-PAGE gels, cryomicrotome slicing of broad gel lanes, and MS data processing. Performance of high resolution profiling is demonstrated for a protein complex preparation from mouse kidney endosome-enriched membranes. Finally, the benefits from increasing resolution and precision of mass spectrometric quantification are discussed.
1. Preparative BN-PAGE
2. Gel sampling and digestion
3. Mass spectrometry
The vast majority of conventional BN-MS studies as well as the recently established high-resolution csBN-MS approach have been applied to mitochondrial and plastid preparations that are (i) readily available, (ii) have limited complexity, and (iii) express target (membrane) protein complexes at high densities. This protocol extends the application of high-resolution complexome profiling to non-mitochondrial membranes expressing low-abundant proteins, about which little information on their integration into complexes is available. For demonstration purposes, we chose an endosome-enriched membrane preparation from mouse kidney obtained by density gradient centrifugation.
Optimization of this preparation has been guided by the marker protein TPC1 that forms intracellular ion channels predominantly localized to early and recycling endosomes12. It is also highly expressed in renal proximal tubular cells, as shown by immunohistochemical analysis of kidney tissue sections (Figure 1A). These membranes were gently solubilized (ComplexioLyte 47 at a low protein:detergent ratio of 1:8) and concentrated on a sucrose cushion by ultracentrifugation. The latter turned out to be an important step for removing excess lower molecular weight components (i.e., detergents, lipids, salts, organic polymers, and metabolites) that tend to negatively impact on resolution of preparative BN-PAGE separations.
Complex separation on a native 1%-13% (w/v) polyacrylamide gradient gel (Figure 1B, middle panel) showed strongly stained protein bands with very little migration artifacts. SDS-PAGE separation of a narrow BN-PAGE gel strip (Figure 1B, frame boxed in red) as a second dimension followed by western blot analysis showed a well-resolved pattern of distinct TPC1-associated complex populations (Figure 1B, upper panel, marked by red arrows), most likely resulting from association with additional protein subunits and/or posttranslational modifications (such as glycosylation12). A 3 cm section of interest was excised, fixed, and processed for cryomicrotome slicing as described11. The individual steps of this procedure (in particular, the precise alignment of the broad gel section), which is of critical importance for preserving resolution during sampling, are documented in the accompanying video. The embedded gel section was finally cut into 101 gel slices with a uniform thickness of 0.25 mm (Figure 1B, lower panel), which were separately digested and analyzed by high performance LC-coupled mass spectrometry.
In addition to size resolution, the quality of protein quantification is key for successful complexome profiling. With the MS set-up and settings used, analysis of the samples was quite comprehensive, resulting in an average identification of more than 1,000 proteins and 10,000 peptides (8,200 of which were protein-specific) per slice, and around 3,000 proteins and 43,000 peptides (38,500 of which were protein-specific) in total. Nevertheless, due to the stochastic nature of data-dependent MS/MS sequencing and its limitations in dynamic range, intensity information was still fragmentary for less abundant proteins. Therefore, an elaborate MS data processing procedure11was performed that is based on precise assignment of peptide signals (peak volumes [PVs] = peptide-related signal intensities integrated over m/z and time) over the entire series of datasets.
As shown in Figure 2A,B, deviations of peptide signals in mass and retention time that remained after calibration were identical for MS-sequenced and for indirectly assigned PVs (with very narrow tolerances of <1 ppm and <0.5 min for 95% of the PVs), indicative of a very low rate for false-positive PV assignment. Remaining outliers were filtered based on their consistency with other related PVs. Since all MS measurements were performed consecutively on the same LC-MS set-up without changes in parameters or hardware components, run-to-run variations (determined as the median of all PV intensities in a sample relative to those in the neighboring slices) were small and easily eliminated by rescaling of the PV datasets (Figure 2C). The resulting peptide intensity information was then used to reconstruct 2,545 protein relative abundance profiles. As shown in Figure 2D, more than 75% of these protein profiles were based on at least three independent protein-specific peptides.
Next, the protocol assessed relevance of the step size of BN-PAGE gel sampling for the resolution of protein complexes. For this purpose, slice datasets were joined by summing the PV information from two, three, or four consecutive slices, thus simulating the outcome for step sizes of 0.5 mm, 0.75 mm, and 1 mm (compared to the original sampling of 0.25 mm). Figure 3 illustrates the resulting abundance profiles for protein TPC1 as an example (A-D). At 0.25 mm, relative intensities and size separation of TPC1-associated complex populations (Figure 3A) were nicely in agreement with the results from western blot analysis (Figure 1B, upper panel); although, the profile showed some noise, mostly resulting from missing values ("gaps") in the PV matrix used for quantification.
Joining of two slices corresponding to 0.5 mm maintained the correct intensities and separation of the TPC1-associated complexes and removed quantification noise (Figure 3B). In contrast, larger step sizes of 0.75 mm and 1 mm (Figure 3C,D) led to a loss of size resolution and abolished discrimination of TPC1 complex subpopulations. It should be noted that the vast majority of published conventional BN-MS analyses use manually cut 2 mm slices (around 60 to cover the entire gel lane)7,8,9,10.
Conversion of migration distance or slice index to molecular size is generally based on markers, either commercially available native standard proteins or well-characterized endogenous protein complexes with known subunit composition (mostly [super]complexes of the mitochondrial oxidative respiratory chain [OXPHOS])13. However, since BN-PAGE separation is based on the effective molecular cross-section that is determined not only by the molecular mass but also by the 3D structure and number of associated lipids, detergent, and Coomassie molecules, individual proteins may show larger deviations. Therefore, it was chosen to use larger sets of protein complexes as markers11. The plot in Figure 4 shows 23 selected markers with a representative subunit shown as a black circle, indicating the log10 values of its predicted molecular mass (according to the UniProtKB/Swiss-Prot database) vs. the slice index of the corresponding profile peak maximum. The latter were obtained from automatized Gaussian fits to the relative abundance data, as shown in the inset of Figure 4 showing the example with chaperone BCS1. Linear regression (red line) provided a function to convert slice index values to apparent molecular sizes, which ranged from 160-630 kDa, along the investigated gel section.
Finally, the analysis provided information on well-characterized complexes and demonstrated the existence of novel subunits and complex assemblies. Examples highlighting different aspects of the complexome are shown in Figure 5 (A-C: proteins expressed or preferably located in endosomal compartments; D-F: complexes from other subcellular localizations). The iron-transporting protein ferritin is known to form complexes from 24 light (FRIL1) and/or heavy (FRIH) subunits, with a total molecular weight of 440 kDa14 (Figure 5A, filled arrow). The subunit profiles (Figure 5A) suggest the existence of at least two smaller forms of the complex (with an apparent mass of 360 kDa and 340 kDa; open arrows) with distinct heavy/light chain stoichiometries (better visible after rescaling of abundances, inset of Figure 5A) that are abundantly present in endosomes.
In contrast, nicalin-nomo1 complexes15 (Figure 5D), the gamma-secretase core complex16 (Figure 5B), and the GPI-transamidase machinery17 (Figure 5E) show fixed abundance ratios of their core subunits over the entire size range and independent from association with additional proteins. This indicates that their subunits are exclusive to each other. Vacuolar H+-ATPases are multiprotein complexes assembled from a pool of more than 20 subunits in a modular manner with a total molecular weight of around 900 kDa. Figure 5C reveals sub-complexes with distinct compositions from at least 17 subunits, either representing biological (dis)assembly intermediates or subcomplexes generated by the experimental conditions, some of which have also been observed in a recent BN-MS study18. Another multi-protein complex example is the proteasome19 (Figure 5F). Close inspection of the abundance profiles of the alpha and beta subunits forming the 20S proteasome core suggests two major complex populations with subtle differences in size (590 kDa and 575 kDa, indicated by grey arrows) and integration of three beta subunits.
In summary, csBN-MS complexome profiling of endosome-enriched kidney membranes provide comprehensive and detailed results regarding the (i) integration of uniform, low-abundant target proteins into complexes, (ii) general complex subunit composition and stoichiometry, and (iii) complex heterogeneities, substructures, and (dis)assembly intermediates.
Figure 1: Preparative BN-PAGE separation of solubilized endosome-enriched membranes from mouse kidney using intracellular channel TPC1 as a marker. (A) Immunohistochemical localization of TPC1 protein in renal proximal tubules by confocal microscopy. Green: anti-TPC1 antibody12 staining visualized with secondary Cy3-biotinylated goat anti-rabbit IgG; red: biotinylated Lotus tetragonolobus lectin (LTL, 10 µg/mL, FITC-conjugated) marking the luminal surface of proximal tubulus cells. The inset shows staining of a corresponding section from a TPC1-KO kidney as a negative control. White scale bars are 20 µm. Also of note is strong TPC1 expression in intracellular vesicles, known from independent experiments to represent early and recycling endosomes12. (B) Preparative BN-PAGE separation of 2.5 mg of solubilized endosome-enriched membranes on a 1%-13% (w/v) polyacrylamide gradient gel. A narrow lane (framed in red) was cut for subsequent SDS-PAGE/western blot analysis (upper panel), resolving different TPC1-associated complexes and glycosylation patterns (red arrows: anti-TPC1/anti-rabbit HRP/ECL prime; green: positions and predicted masses [MDa] of marker protein complexes identified by total protein staining [SYPRO Ruby blot stain]) of the blot. From right to left: Na+/K+-transporting ATPase, Cytochrome b-c1 complex dimer, ATP synthase, NADH:ubiquinone oxidoreductase. A 3 cm section of interest from the gel lane was excised, embedded in tissue embedding medium, mounted, and sliced into 101 sections (0.25 mm) along the protein migration front using a cryomicrotome (lower panel; see video link). Please click here to view a larger version of this figure.
Figure 2: Key parameters determining accuracy of MS signal assignment and quantification as well as depth of analysis. (A) Distribution of relative mass errors (in ppm) after m/z calibration of MS/MS sequenced (red bars) and indirectly assigned (i.e., based on closely matching mass and retention times, see protocol; blue bars) peptide signals. This suggests a final mass error of <1 ppm (for 95% of the signals/assigned PVs) and very low rate of false positive assignments. (B) Distribution of nano-HPLC retention time deviations from total average after elution time alignment of peptide signals, using Loess regression (see protocol) and color coding as used in (A). Time error is less than 30 s for >95% of peptide signals/assigned PVs. (C) Run-to-run variation of total MS intensities plotted relative to the average of the two neighboring samples. These scale factors were applied to the raw PV tables to minimize systematic technical errors. (D) Peptide information used for calculating relative abundance profiles of proteins. After filtering protein-specific peptides for outliers, poorly scored, or single identifications (see protocol), 2,545 protein abundance profiles were determined, >75% of which were based on at least three peptides with reasonable confidence. Please click here to view a larger version of this figure.
Figure 3: Critical impact of step size in gel sampling on complexome resolution of TPC1. Datasets were joined by summing the signal intensities in groups of 1, 2, 3, and 4 consecutive slices (A-D, respectively) and processed identically to simulate different step sizes in gel slicing as indicated. The TPC1 profile shows some (oversampling) noise at 0.25 mm but good size resolution of three complex populations (see also Figure 1B), which is largely preserved at a 0.5 mm step width. Discrimination of these populations becomes lost as 1 mm is approached. Please click here to view a larger version of this figure.
Figure 4: Determination of apparent molecular weight. 23 marker complexes with defined molecular composition (as indicated, according to UniProtKB/Swiss-Prot) were used as size markers. Logarithmic values of their expected molecular weights (in kDa) were plotted vs. the profile peak maximum slice index of the indicated representative protein subunit (filled circles in black). Linear regression fitting to this data (red line) provided a function converting slice index values to apparent molecular weights. Peak maxima were determined by automatized Gaussian fits to protein profile peaks as shown in the inset (right) for the chaperone protein BCS1 (primary data in blue, fit boundaries indicated by orange lines, fit function in red). In addition, these fits determined peak half-maximal widths (green line, 6.5 slices, or 1.6 mm for the example shown) with the sharpest focusing complexes spanning around a 1.5 mm gel. Please click here to view a larger version of this figure.
Figure 5: Examples of protein complex subunit profiles. Relative protein abundance vs. apparent molecular weight plotted for heavy and light chain of ferritin (A) revealing molecular heterogeneity of ferritin subunit stoichiometry, more clearly visible after rescaling of abundances (inset). Filled arrow and open arrows denote the full complex (440 kDa) and two subcomplexes, respectively. The gamma-secretase (B) subunits quantitatively integrated into a single-core complex population. The subcomplexes of vacuolar H+-ATPases (C) exhibited multiple assemblies with distinct subunit composition, all expressed in endosomes. The nomo1 and nicalin proteins (D) formed an exclusive complex (the GPI-transamidase), which is a multi-subunit enzymatic machinery forming several complexes. (E) The 20S proteasome core complex showing (F) a subtle subcomplex pattern with two populations indicated by arrows in grey, all originating from other subcellular localizations. Please click here to view a larger version of this figure.
The presented study built on the csBN-MS technique previously benchmarked with a mitochondrial preparation11 and incorporated improvements in sample preparation, gel processing, and MS data evaluation. Focused analysis of a section of the large-scale separation BN-PAGE gel provided a comprehensive set of data showing quality measures comparable to the study with mitochondrial membranes. Mass and retention time errors as well as run-to-run variations were kept very low and provided the basis for determining reliable protein abundance profiles. Size resolution appeared to be good, with half-maximal peak widths as low as six slices (corresponding to 1.5 mm, Figure 4) and relative size differences of less than 10% resolved (Figure 3, Figure 5A). These values did not fully meet the size resolution quality of the previous csBN-MS analysis of mitochondria (despite the smaller gel sampling step size chosen), but they are significantly better than the performance of conventional BN-MS or size-exclusion MS approaches20 that have recently become popular.
The importance of a high effective complex size resolution is underlined by the simulation experiment in Figure 3 (using the TPC1-associated complexes) that can hardly be resolved by 2D BN/SDS-PAGE western blot analysis (Figure 1B). These results suggest that the 0.25 mm slicing in this case resulted in some oversampling, but it this still proved to be useful for elimination of "quantification noise" without compromising effective size resolution. Thus, in line with previous results11, a sampling step size of ~0.3 mm is generally recommendable.
Notably, discrimination of TPC1-associated complexes is completely lost with 1 mm gel sampling, which is the smallest step size provided by manual slicing in conventional BN-MS5,6. This may explain the fact that despite powerful MS technologies being available, very few protein complexes and subunits have been identified de novo by complexome profiling. Besides its good resolving power, csBN-MS offers high versatility. Membrane-bound complexes and soluble protein complexes ranging from 50 kDa to several MDa can be effectively resolved in a single experiment with minimum bias11. This contrasts with alternative separation techniques used for complexome profiling like size exclusion or ion exchange chromatography, which operate with subsets of soluble proteins with certain size ranges or charge properties. On the downside, csBN-MS is less scalable (maximum load of ~3 mg protein per gel), may be technically challenging, and cannot be automatized.
Overall, the results demonstrate that csBN-MS-based complexome profiling can be successfully applied to non-mitochondrial targets but also indicate some associated challenges. Thus, efficient extraction and biochemical stability of protein complexes require more optimization, and cleaning steps and may still be limited. Within the investigated size window, the number of well-focused, monodisperse protein complexes was indeed considerably lower (data not shown) compared to a mitochondrial sample. It is also recommended to lower BN-PAGE sample loads to obtain acceptable gel separation. Higher loads may require broader gel lanes that are more difficult to process properly for slicing (see accompanying video). Furthermore, protein complexity of the samples was higher (around two-fold) than the mitochondria-derived slice digests, leading to more missing PV values and a reduced dynamic range. In fact, some small proteins expected to be part of the complexes shown in Figure 5 were missing in the analyses. These problems can be resolved in the future by using faster and more sensitive MS instruments or data-independent acquisition modes.
Sample preparation is highly critical for protein complex retrieval, stability, and gel separation quality. Parameters and procedures should be optimized for each source tissue, cell lysate, membrane (fraction), and protein complex of interest. The following general recommendations are provided that may help extend applications of csBN-MS:
(i) Preparing samples fresh and avoiding warming/freezing, strong dilutions, changes in buffer conditions, and unnecessary delays;
(ii) Using buffers that are essentially devoid of salts (replace with 500-750 mM betaine or aminocaproic acid), about a neutral pH, and containing up to 1% (w/v) of non-denaturing detergent (protein:detergent ratio between 1:4-1:10 for solubilization of membrane protein complexes, no detergent required for soluble protein complexes);
(iii) Careful testing and adjusting of detergent conditions by analytical BN-PAGE, since these may strongly impact efficiency of complex solubilization, representation of membrane protein complexes in the sample, stability, and homogeneity of protein-detergent micelles. The latter are prerequisites for proteins to focus as distinct bands/complex populations on BN-PAGE gels. Previous literature offers a broad range of neutral detergents. However, DDM (n-dodecyl β-d-maltoside)1,2,4,5,6 and digitonin3,5,7,8,9,10,13,18 have been the most popular choices for BN-MS analyses so far. It must be emphasized that any detergent condition necessarily represents a compromise between solubilization efficiency and preservation of protein interactions and may not be equally suited for all types of target protein and source material;
(iv) Removing charged polymers like fibrils, filaments, polylysine, DNA, and abundant lower molecular weight components (i.e., metabolites, lipids, or peptides). This may be accomplished by ultracentrifugation, gel filtration, or dialysis. This is particularly important for total cell or tissue lysates;
(v) Adding Coomassie G-250 (final concentration 0.05%-0.1%) and sucrose (to increase density for loading, final concentration 10%-20% [w/v]) to the sample just prior to loading, to clear by short ultracentrifugation, load the sample without perturbation, and start the run immediately thereafter.
As a future perspective, csBN-MS-based complexome profiling offers options for multiplexing to study protein complex dynamics or changes related to specific biological conditions. Combined separation of metabolically labelled samples as proposed for size-exclusion based profiling21 appears straightforward, but it may be hampered by spontaneous subunit exchange in complexes occurring independently of the used separation method. Alternatively, labelled samples can be resolved in neighboring gel lanes, which can then be co-sliced or combined post-digesting for differential analysis with high sensitivity and robustness.
The authors have nothing to disclose.
This study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-ID 403222702 – SFB 1381 and under Germany's Excellence Strategy CIBSS – EXC-2189 – Project ID 390939984. We thank Katja Zappe for technical assistance.
30% Acrylamide/Bis Solution, 37.5:1 | Bio Rad | #1610158 | Recommended for acrylamide gradient gel solutions up to 13% |
30% Acrylamide/Bis Solution, 19:1 | Bio Rad | #1610154 | Recommended for acrylamide gradient gel solutions >13% |
SYPRO Ruby Protein Blot Stain | Bio Rad | #1703127 | Total protein stain on blot membranes; sensitive and compatible with immunodetection |
Coomassie Brilliant Blue G-250 | Serva | no. 35050 | Centrifugate stock solutions prior to use |
ComplexioLyte 47 | Logopharm | CL-47-01 | Ready-to-use detergent buffer (1%) for mild solubilization of membrane proteins |
Embedding Medium / Tissue Freezing Medium | Leica Biosystems | 14020108926 | Embedding medium for gel sections to be sliced by a cryo-microtome |
Immobilon-P Membrane, PVDF, 0,45 µm | Merck | IPVH00010 | |
ECL Prime Western Blotting Detection Reagent | GE Healthcare | RPN2232 | |
Plastic syringe with rubber stopper, 20-30 ml | n.a. | n.a. | any supplier, important for making gel section embedding tool |
broad razor blade | n.a. | n.a. | any supplier, for BN-PAGE gel trimming / excision of lanes |
metal tube / cylinder, ca. 4 cm long | n.a. | n.a. | mold for embedding and freezing of gel samples |
Protein LoBind Tubes, 1.5 ml | Eppendorf | Nr. 0030108116 | highly recommended to minimize protein/peptide loss due to absorption |
sequencing-grade modified trypsin | Promega | V5111 | |
C18 PepMap100 precolumn, particle size 5 µm | Dionex / Thermo Scientific | P/N 160454 | |
PicoTip emitter (i.d. 75 µm; tip 8 µm) | New Objective | FS360-75-8 | |
ReproSil-Pur 120 ODS-3 (C18, 3 µm) | Dr. Maisch GmbH | r13.93. | columns packed manually |
rabbit anti-TPC1 antibody | Gramsch Laboratories | custom production | described in Castonguay, et al., 2017 (Reference 12) |
Cy3-biotinylated goat anti-rabbit IgG | Vector Laboratories | CY-1300 | described in Castonguay, et al., 2017 (Reference 12) |
biotinylated Lotus tetragonolobus lectin, FITC-conjugated | Vector Laboratories | #B1325 | described in Castonguay, et al., 2017 (Reference 12) |
cryo-microtome Leica CM1950 | Leica Biosystems | 14047743905 | |
Mini Protean II Cell with wetblot unit | Bio Rad | n.a. | for SDS-PAGE and Westernblot (not sold any more) |
Penguin Midi Gel Electrophoresis System | PeqLab | n.a. | for BN-PAGE (not sold any more) |
Zeiss Axiovert 200 M microscope + Photometrics Coolsnap 2 digital camera | Zeiss / Photometrics | n.a. | |
peristaltic pump (IP high precision multichannel) | Ismatec | ISM940 | for casting of gradient polyacrylamide gels |
gradient mixer with stirring (two chambers) | selfmade, alternatively Bio Rad | 1652000 or 1652001 | for casting of gradient polyacrylamide gels, manual provides instructions to cast linear or hyperbolic gradient gels (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1652000.pdf) |
ultracentrifuge Sorvall M120 with S80 AT3 rotor | Sorvall / Thermo Scientific | n.a. | for sample preparation (not sold any more) |
UltiMate 3000 RSLCnano HPLC | Dionex / Thermo Scientific | ULTIM3000RSLCNANO | |
Orbitrap Elite mass spectrometer | Thermo Scientific | IQLAAEGAAPFADBMAZQ |