A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

Podsumowanie

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

Streszczenie

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (¹⁴N/¹⁵N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

Wprowadzenie

Photosynthetic processes in thylakoid membranes of plants and algae can function in a linear and cyclic mode. During linear electron flow (LEF) photosystem I (PSI), photosystem II (PSII) and cytochrome b₆/f ultimately transfer electrons from water to NADP+¹, leading to the generation of NADPH and ATP². In contrast, cyclic electron flow (CEF), which is known to be induced under diverse environmental conditions like state 2 ³ and anaerobic conditions⁴, results in the re-reduction of oxidized PSI by injecting electrons back into the electron transport chain. This process can take place either at the stromal side of the cytochrome b₆/f complex¹ or at the plastoquinone pool⁵ and generates ATP, but no NADPH².

The aim of the presented protocol is to demonstrate a mass spectrometry (MS) based method for the comparative, quantitative analysis of multiprotein complexes in thylakoid membranes of Chlamydomonas reinhardtii to gain insight into the composition of these complexes under different conditions (exemplified by comparing genetically different strains). This approach was applied in a publication by Terashima et al. in 2012 showing a Ca²⁺-dependent regulation of CEF in C. reinhardtii mediated by a multiprotein complex including the proteins CAS, ANR1, and PGRL1⁶. The procedure will be explained by comparatively analyzing the composition of the CEF-supercomplex in two genetically different strains, thereby taking advantage of labeling one of the two strains with heavy nitrogen (¹⁵N). Briefly, the protocol includes the preparation of thylakoid membranes, followed by detergent solubilization and fractionation of photosynthetic complexes in a sucrose density gradient. After fractionation of the gradient, selected fractions of two strains are mixed based on equal volume, separated by SDS-PAGE followed by in-gel digestion and subsequent quantitative MS analysis.

As mentioned above, CEF is induced under different environmental conditions and a publication from 2010 demonstrates the isolation of a functional CEF-supercomplex from state 2 locked cells of C. reinhardtii⁷, which was performed by separating solubilized thylakoid membranes on a sucrose density gradient during ultracentrifugation. Different from Iwai et al.⁷, the presented protocol describes the isolation of the CEF-supercomplex from anaerobic grown C. reinhardtii cultures by following an alternative procedure. This comprises changes in the thylakoid isolation protocol as well as differences concerning the solubilization step and the separation of protein complexes by ultracentrifugation. In the present protocol, thylakoid membranes are isolated by applying the procedure published by Chua and Bennoun⁸, while the buffers used for thylakoid preparation by Iwai et al. contained 25 mM Mes, 0.33 M sucrose, 5 mM MgCl₂, 1.5 mM NaCl (pH 6.5) as described⁹. The solubilization was performed with 0.7-0.8% detergent (n-tridecyl-β-D-maltoside) for 30 min on ice in the case of Iwai and coworkers, while the solubilization method described here relies on the use of 0.9% detergent (n-Dodecyl-β-D-maltoside (β-DM)) and is performed for only 20 min on ice. Both groups used 0.8 mg of chlorophyll per ml for the solubilization with the respective detergent. For the separation of photosynthetic complexes from solubilized thylakoid membranes Iwai et al. applied sucrose concentrations between 0.1-1.3 M, whereas the authors of this protocol used concentrations ranging from 0.4-1.3 M. The last difference is the centrifugation speed, which is lower compared to the earlier publication.

Solubilization of thylakoid membranes with nonionic detergents followed by sucrose density gradient fractionation has already been applied in numerous studies ranging from the 1980s until today^{7, 9-14} and also the application of metabolic labeling of proteins is a widespread method in the proteomics field. The described approach applies the ¹⁵N metabolic labeling for one of the two compared strains by culturing it in the presence of heavy nitrogen as sole nitrogen source in the form of ¹⁵N NH₄Cl, which is incorporated into all amino acids leading to a mass shift depending on the amino acid sequence of the peptide. When analyzing a mixture of ¹⁴N and ¹⁵N within one MS run, this mass shift can be used to determine the sample origin for each peptide and relative peptide abundances can be calculated representing relative abundances for the corresponding protein¹⁵.

Numerous quantitative proteomics studies on C. reinhardtii are available, which compare a defined amount of protein to analyze changes in the proteome between experimental conditions (e.g. changes in the proteome due to nutrient^16-19 or light stress^20,21). Compared to those studies, in the currently presented approach equal volumes of samples are combined and analyzed. This setup allows to study the migration behavior of proteins within the gradient and moreover to analyze the composition of different complexes with respect to the investigated strains.

This method will be explained by mainly concentrating on three proteins: The first candidate is the chloroplast-localized calcium sensor protein CAS, which was shown to be involved in photo-acclimation in C. reinhardtii²². Calcium is considered to be an important signaling ion for pathways that are activated due to different biotic and abiotic stresses finally leading to changes in gene expression and cell physiology²³ and it was proposed that chloroplasts might contribute to cellular Ca²⁺ signaling via the CAS protein^22,24,25. The second protein is ANR1 (anaerobic response 1 ⁶), a protein that was shown to be induced under anoxic growing conditions in C. reinhardtii²⁶. Notably, CAS as well as ANR1 were identified as subunits of the CEF-supercomplex and moreover, by using reverse genetic approaches, it was demonstrated that both proteins contribute functionally to CEF in vivo⁶, supporting their role as functional subunits of this protein complex. The third protein is the thylakoid protein PGR5-Like 1 (PGRL1), which was shown to be involved in CEF in Chlamydomonas^4,27  as well as in Arabidopsis^5,28  and was also identified in the work of Iwai et al.⁷ 

This approach will be presented by showing the results of two different experiments: wildtype (WT) versus (vs.) a ΔPSI²⁹ strain, exhibiting a deletion of the psab gene, coding for an essential photosystem I subunit, which is also part of the CEF-supercomplex and WT vs. a pgrl1 knock-out strain⁴. For each of those experiments the quantitative composition of the CEF-supercomplex between a ¹⁵N- and a ¹⁴N-labeled strain has been compared.

Protokół

1. Culturing of Chlamydomonas

1. The following C. reinhardtii strains were used in the present study: WT cc124, WT cw15-arg7 (cell-wall deficient and arginine auxotroph), a ΔPSI mutant strain²⁹ and a pgrl1 knock-out strain⁴.
2. All strains were grown in Tris-Acetate-Phosphate (TAP)-medium³⁰, at 25 °C with a continuous light intensity of 20-50 µE/m² sec and shaking at 120 rpm. The culture of ΔPSI should be wrapped with some tissue paper for light exposure of <5 µE/m² sec.
3. The cultures of the labeled strains (ΔPSI and WT cc124, respectively) contained 7.5 mM ¹⁵N NH₄Cl, whereas the cultures for the nonlabeled strains (WT cw15-arg7 and pgrl1, respectively) contained 7.5 mM ¹⁴N NH₄Cl. Cells have to grow for at least four generations to achieve full labeling and must be kept at exponential growth phase.
4. On the day before starting the experiment count and dilute cells to a density of 1 x 10⁶ cells/ml in a volume of at least 750 ml/strain.

Please note that two types of discontinuous sucrose density gradients are described in the following protocol. The photosystem sucrose density gradients according to Takahashi et al.⁹ are used to separate the different photosynthetic protein complexes from isolated and solubilized thylakoids during over-night centrifugation and have to be prepared the day before (see Protocol 2) and the thylakoid sucrose density gradients⁸ are applied in the thylakoid isolation procedure (see Protocol 3).

2. Preparation of Photosystem Sucrose Density Gradients

1. For pouring the gradients three stock solutions are needed:
   1. 2 M Sucrose
   2. 10% β-DM in H₂O
   3. 0.5 M Tricine, pH 8.0 (NaOH)
2. Using these stocks, prepare the following solutions:

   Concentration:	1.3 M	1.0 M	0.85 M	0.7 M	0.65 M	0.4 M
   2 M Sucrose	13 ml	10 ml	8.5 ml	7 ml	6.5 ml	4 ml
   10% β-DM	100 µl	100 µl	100 µl	100 µl	100 µl	100 µl
   0.5 M Tricine	200 µl	200 µl	200 µl	200 µl	200 µl	200 µl
   H2O	6.7 ml	9.7 ml	11.2 ml	12.7 ml	13.2 ml	15.7 ml

3. Use 14 mm x 89 mm centrifuge tubes and pour the gradients very slowly, starting with the highest sucrose density solution to lower density solution.
4. Pour only 1 ml each for the 1.3 and 1.0 M solutions and 2 ml for the rest of the solutions.
5. Leave gradients overnight in the cold room.

3. Anaerobic Induction and Isolation of Thylakoid Membranes⁸

1. Before starting with the isolation of thylakoid membranes, induce anaerobic conditions by bubbling with argon for 4 hr. The bubbling can be performed through a glass pipette in culturing flasks with constant mixing of the culture with a magnetic stir bar.
2. Start with the isolation of thylakoid membranes by pelleting the cells for 5 min at 2,500 x g, resuspend in H1 buffer (0.3 M Sucrose, 25 mM HEPES (pH 7.5), 5 mM MgCl₂).
   (Please note that when starting with the isolation of thylakoid membranes samples should be kept on ice to avoid protein degradation, all centrifugation steps are carried out at 4 °C and working with gloves is strongly recommended to avoid keratin contamination.)
3. Pellet cells for 5 min at 2,500 x g, resuspend in H1 buffer.
4. Break cells with two passages through a nebulizer with a nitrogen pressure of 1,500 hPa (for strains without cell wall do only one passage).
5. Spin down cells for 7 min at 2,500 x g.
   (After this step the supernatant should be light green, if not, the cell breakage was not successful.)
6. Resuspend cells in H2 buffer (0.3 M Sucrose, 5 mM HEPES (pH 7.5), 10 mM EDTA (pH 8.0)) and pellet cells for 10 min at 32,800 x g.
7. Resuspend cells in H3 buffer (1.8 M Sucrose, 5 mM HEPES (pH 7.5), 10 mM EDTA (pH 8.0)) and homogenize pellet with a potter (no green particles should remain at the end of this working step).
8. Prepare thylakoid sucrose density gradients in tubes (25 mm x 89 mm) by starting at the bottom with a layer of 12 ml H3 buffer with cells, pour a middle layer of 12 ml H4 buffer (1.3 M Sucrose, 5 mM HEPES (pH 7.5), 10 mM EDTA (pH 8.0)) and on top 12 ml H5 buffer (0.5 M Sucrose, 5 mM HEPES (pH 7.5), 10 mM EDTA (pH 8.0)). Be careful when pouring the gradients and avoid mixing of the three layers.
9. Balance with H5 buffer and centrifuge thylakoid sucrose density gradients for 1 hr at 70,700 x g.
10. Remove thylakoid bands from the thylakoid sucrose density gradients and dilute them with an appropriate volume H6 buffer (5 mM HEPES (pH 7.5) 10 mM EDTA (pH 8.0)).
11. Spin down cells at 37,900 x g for 20 min. If the pellet is not tight, more H6 buffer is required for this centrifugation step.
12. Resuspend thylakoids in a small volume H6 buffer and proceed with determination of the chlorophyll concentration.

4. Determination of Chlorophyll Concentration³¹

1. The determination of the chlorophyll amount is performed with 80% acetone.
2. Mix 995 µl 80% acetone and 5 µl of thylakoids in H6 buffer (dilution 1:200).
3. Vortex for several seconds until no green particles remain and then spin down at 14.1 x g for 5 min.
4. Take the supernatant and measure the extinction at 663.6 and 646.6 nm and 750 nm, respectively.
5. Calculate the chlorophyll amount using the following formulas:
   1. C_{Chl a} [mg/ml] = (0.01225 * E_663.6 – 0.00255 * E_646,6) * dilution factor
   2. C_{Chl b} [mg/ml] = (0.02031 * E_646.6 – 0.00491 * E_663.6) * dilution factor
   3. C_Chl [mg/ml] = C_{Chl a} + C_{Chl b}

5. Loading of Photosystem Sucrose Density Gradients

1. Calculate the amounts of thylakoids, β-DM and H6 buffer that are needed for each gradient:
   1. Each gradient should be loaded with a total volume of 700 µl with 0.8 mg/ml chlorophyll in 0.9% β-DM (dissolve β-DM in H₂O), fill the remaining volume with H6 buffer.
2. For solubilization prepare different aliquots with thylakoids, β-DM and H6 buffer for each gradient.
3. Leave samples for 20 min on ice with regularly mixing (inverting) every few minutes (solubilization step).
4. Centrifuge at maximum speed (14,000 x g) for 10 min at 4 °C.
   (After centrifugation, the pellet should be small and whitish while the supernatant is dark-green.)
5. Load supernatant on the gradients.
6. Balance with H6 buffer and spin using ultracentrifuge at 134,470 x g overnight (14 hr) at 4 °C.
   (Please note that the successful separation of photosynthetic complexes using photosystem sucrose density gradients as presented in this protocol is only achieved when using freshly isolated thylakoids, since a prediction for solubilization and complex separation is difficult to make when working with thylakoid membranes that have been frozen before.)

6. Fractionation of Photosystem Sucrose Density Gradients

1. Take pictures of the gradients.
2. Puncture a hole at the bottom of the tube using a needle and fractionate gradients in 500 µl tubes. Alternatively, the fractionation can also be performed by using a 96-well plate (microplate).
3. When using a microplate, determine absorbance of the different gradient fractions at 675 nm.
   (At this step samples can be stored at -80 °C for a few weeks.)

7. SDS-PAGE and Immunodetection

(Please note that only for the comparison of WT vs. ΔPSI a western blot analysis has been performed to select the fractions for subsequent MS-analysis. For the experiment WT vs. pgrl1 fractions were chosen by means of the absorbance in the different fractions.)

1. Separate 30 µl of each fraction from WT and ΔPSI gradient on a 13% SDS polyacrylamide gel³².
2. Perform western blot analysis³³ using the following antibodies: ANR1 (TEF7)²⁶, PGRL1³⁴, CAS⁶, the PSI subunit PSAD³² and the PSII core subunit D1. Use all antibodies with a dilution of 1:1,000 (for enhanced chemiluminescence detection technique), except for the D1 antibody, which should be used with a dilution of 1:10,000.
3. Based on the results of the immunodetection, take the peak fractions of ANR1, PGRL1 and PSAD for WT and ΔPSI (here fractions 6 and 13, respectively), mix 30 µl of fraction 6 from WT and ΔPSI and do the same with fraction 13 from both preparations and separate them again on a 13% SDS polyacrylamide gel.
4. For the quantitative comparison of WT vs. pgrl1 take the CEF-supercomplex fractions as determined by measuring the absorbance in the different gradient fractions and mix 30 µl of both samples followed by separation on a 13% SDS polyacrylamide gel.
5. Stain the protein bands with Coomassie solution (85% phosphorous acid, 757 mM ammonium sulfate, 1.2% Coomassie brilliant blue, 20% methanol) for 2 hr at room temperature or over-night at 4 °C.
6. To remove unspecific staining, wash several times with ddH₂O.
7. Cut the lanes into 1 mm gel pieces and proceed with the in-gel digestion.
   (Alternatively, samples can be dried a few minutes in a vacuum centrifuge and stored for a few weeks before continuing with the in-gel digestion.)

8. In-gel Digestion (Modified from Shevchenko et al.³⁵)

Please note to prepare all buffers and solutions shortly before use and take glass bottles for buffers containing acetonitrile (ACN).
CAUTION! Working with ACN might be harmful; more information can be downloaded from: http://www.sciencelab.com/msds.php?msdsId=9927335 (2013).

1. Wash gel pieces with >10 volumes of ddH₂O (~200 µl) for 30 sec.
2. Incubate the gel pieces with 300 µl of 25 mM NH₄HCO₃ for 15 min with shaking, remove liquid.
3. Incubate the gel pieces with 300 µl of 25 mM NH₄HCO₃ in 50% ACN (prepare by mixing ACN and 50 mM NH₄HCO₃ at a ratio of 1:1) for 15 min with shaking, remove liquid.
4. If the gel pieces are still blue, repeat the NH₄HCO₃ and NH₄HCO₃ / ACN washes until most of the Coomassie is removed.
   (Although the bulk of Coomassie staining should be removed, it is not necessary to destain the gel pieces completely.)
5. Add 100 µl of ACN to dehydrate the gel pieces for 5 min. The gel pieces should shrink and look completely white.
6. Remove as much ACN as possible and proceed with trypsin digestion. Alternatively, samples can be stored at -20 °C for a few weeks.
7. Add 10-20 µl per band of 20 ng/µl trypsin in 10% ACN / 25 mM NH₄HCO₃ (freshly diluted), keep samples on ice.
8. After 30 min, check if all solution was absorbed and add more trypsin buffer, if necessary. Gel pieces should be completely covered with trypsin buffer. Keep samples on ice.
9. Leave gel pieces for another 90 min on ice to saturate them with trypsin.
   (Critical step: the yield of tryptic peptides increases considerably with increasing incubation times on ice, probably because of slow diffusion of the enzyme into the polyacrylamide matrix. It is necessary to have a small excess of solution covering the gel pieces to avoid that the pieces fall dry during the overnight incubation.)
10. Digest at 37 °C for 4-6 hr. For experiments requiring maximal peptide recovery, digest overnight.
11. After digestion spin down condensed buffer.
12. Sonicate tubes for 5 min to elute peptides and centrifuge again.
13. Collect supernatant and transfer it to a new tube.
14. Perform peptide extraction with 80 µl of 30% ACN / 1% formic acid (FA) in a sonic bath for 15 min.
15. Perform extraction with 80 µl of 30% ACN / 1% FA in a sonic bath for 15 min.
16. Perform extraction with 80 µl of 70% ACN / 1% FA in a sonic bath for 15 min.
17. Centrifuge again and pool supernatant with the prior eluate.
    (FA serves to inactivate trypsin and increase peptide solubility.)
18. Dry peptide solution completely in a vacuum centrifuge.
    (At this point samples can be stored for a few weeks in -20 °C before proceeding with MS-analysis.)
19. Resuspend peptides in 6 µl MS buffer (5% ACN, 0.1 v/v FA, water) and sonicate for 2-5 min.
20. Centrifuge samples at maximum speed for 5 min to remove particulate material.
21. Use 4 µl of supernatant for mass spectrometric analysis.

9. MS-Data Analysis with “Proteomatic”

The data analysis was performed with the open-source software “Proteomatic” (which can be downloaded at http://www.proteomatic.org/), a platform that allows the generation and completion of MS/MS data evaluation pipelines, by using free and commercial software³⁶. Briefly, the settings are described below and more detailed information can be found in^6,26.

1. Identification and Quantification of MS-data
   Identification and quantification of proteins from the experiments WT vs. ΔPSI were done with OMSSA (version 2.1.4³⁷) and qTrace²⁶, respectively, as described^6,26. For the MS-data analysis from the experiment WT vs. pgrl1 the following updated pipeline was used:
   1. In addition to OMSSA³⁷, proteins were also identified with X! Tandem (version 2013.02.01³⁸). Both algorithms were applied by searching against a database generated by combining the JGI Chlamydomonas gene model database version 4.3 with the AUGUSTUS database version 10.2 as well as against a joined database of the NCBI databases BK000554.2 and NC_001638.1.
   2. MS² identification of peptides was performed by using a target-decoy approach³⁹. A decoy for each protein was created by randomly shuffling tryptic peptides while retaining the redundancy of nonproteotypic peptides.
   3. Statistical validation of the peptides was performed with the software qvality (version 0.3.3⁴⁰) using a posterior error probability (PEP) threshold of less than 0.01 and results were filtered with a precursor mass tolerance of 5 ppm.
   4. Peptides from OMSSA and X! Tandem runs were joined and quantified with qTrace²⁶ applying the following filter steps: require MS² identification, add protein names / group information and require both sister peptides.

To investigate whether protein ratios were significantly different towards each other in the mixed CEF-supercomplex fractions from WT and pgrl1, statistical analysis was performed applying the software SPSS (version 21). The subunits of the cytochrome b₆/f complex were tested against each other as well as the proteins FNR, FTSH2 and HCF136 against the PSI subunits. The peptide ratios of each scan count per protein of all four replicates were tested for normal distribution with the Shapiro-Wilks test. Since the peptide populations were not normally distributed (p<0.001), nonparametric statistics were performed. First, the Kruskal-Wallis test was applied assessing a significant divergence among groups. Only if this test predicted significant differences between groups, further analysis was performed to predict significant differences between two independent groups with the Mann-Whitney-U Test.

Wyniki

The introduced quantitative proteomics approach aims to characterize the composition of multiprotein complexes in thylakoid membranes demonstrated by the comparative analysis of CEF-supercomplex components in genetically different C. reinhardtii strains. The described method has successfully been applied by Terashima et al.⁶ and comprises the isolation of thylakoid membranes from anaerobic grown cultures, followed by detergent solubilization. Subsequently, samples are loaded onto a sucrose de...

Dyskusje

Different quantitative proteomic studies using stable isotope labeling have been published in the last years. In these experiments usually two different samples are compared, of which one sample is labeled with a stable isotope. Thereafter proteins or peptides from the two samples are combined in an equal ratio and further processed together⁴⁸. Such studies often intend to compare defined isolated cellular compartments (e.g. chloroplasts, mitochondria, or thylakoid membranes) exposed to different stre...

Materiały

Name	Company	Catalog Number	Comments
Chemicals
Acetic acid	AppliChem	A0662	http://www.applichem.com/home/
Acetone	AppliChem	A2300	http://www.applichem.com/home/
Acetonitrile Optigrade für LC-MS	Diagonal	9340	Harmful, work with gloves. See protocol text for further precautions. https://www.diagonal.de/
Ammonium chloride 15N	Cambridge Isotope Laboratories	39466-62-1	http://www.isotope.com/cil/index.cfm
Ammonium chloride 14N	AppliChem	A0988	http://www.applichem.com/home/
Ammonium hydrogen phosphate	AppliChem	A3583	http://www.applichem.com/home/
Ammonium sulfate	AppliChem	A3598	http://www.applichem.com/home/
Coomassie brilliant blue R-250	Fisher Scientific	10041653	http://www.de.fishersci.com/index.php/deindex
n-Dodecyl-β-D-maltoside	AppliChem	A0819	http://www.applichem.com/home/
EDTA	AppliChem	A2937	http://www.applichem.com/home/
Formic acid	AppliChem	A3858	http://www.applichem.com/home/
HEPES	AppliChem	A3724	http://www.applichem.com/home/
Magnesium chloride	AppliChem	A4425	http://www.applichem.com/home/
Methanol	AppliChem	A2954	http://www.applichem.com/home/
Phosphorous acid	AppliChem	A0989	http://www.applichem.com/home/
Dipotassium hydrogen phosphate	AppliChem	A1042	http://www.applichem.com/home/
Potassium dihydrogen phosphate	AppliChem	A1043	http://www.applichem.com/home/
Sodium hydroxide	AppliChem	A1551	http://www.applichem.com/home/
Sucrose	AppliChem	A1125	http://www.applichem.com/home/
Tricine	AppliChem	A3954	http://www.applichem.com/home/
Tris	AppliChem	A2264	http://www.applichem.com/home/
Trypsin (sequencing grade modified) and Trypsin buffer	Promega	V5111	http://www.promega.de/
Equipment
Nebulizer (BioNeb cell disruptor)	Glas-Col		http://www.glascol.com/product/subproduct/id/75
Centrifuge tubes (14 mm x 89 mm)	Beckman Coulter	331372	for preparation of Takahashi style gradients http://www.beckmancoulter.de/
Centrifuge tubes 25 mm x 89 mm	Beckman Coulter	344058	for preparation of thylakoid isolation gradients. http://www.beckmancoulter.de/
Coulter Avanti Centrifuge J-20 XP	Beckman Coulter		http://www.beckmancoulter.de/
Fuchs-Rosenthal cell couting chamber	Diagonal	449/72	https://www.diagonal.de/
Homogenizer (Potter) 50 ml	Fisherbrand	10618242	http://www.de.fishersci.com/index.php/defisherbrand
Pistil for homogenizer	Fisherbrand	105252220	http://www.de.fishersci.com/index.php/defisherbrand
Ultracentrifuge (Optima XPN-80 Ultracentrifuge)	Beckman Coulter		website: http://www.beckmancoulter.de/
Other
Antibodies	Agrisera		http://www.agrisera.com/en/index.html