We thank Rana Adeel Ahmad for assistance in FCP purification. Prof. Claudia B&#252;chel is acknowledged for helpful discussions and reading the manuscript. This work was supported by the German Research Foundation to LD (DI1956-1/1) and the Humboldt foundation for a Feodor-Lynen fellowship to LD.

Note: Photosynthetic complexes such as FCPs are highly vulnerable to light and heat. Always work on ice and under a very dim light.
1. Isolation of FCP from Cells
<ol>
	<li>Thylakoid isolation from C. meneghiniana cells
	<ol>
		<li>Grow C. meneghiniana in five 500 mL flasks each filled with 300 mL of ASP-medium23,25 and 50 million cells. Plug the flasks with a cotton stopper and allow the cells to grow to the exponential growth phase for approximately one week on a shaker at 120 rpm with a 16 h light and 8 h dark phase at 40 &#181;mol photons/(m&#178;s) white light and a temperature between 15-18 &#176;C. Check that the cell number is between 1.5-2 million cells/mL with a cell counter chamber.</li>
		<li>Centrifuge the cells at 4,000 x g in a precooled rotor with 500 mL centrifuge vials (4 &#176;C) for 15 min in a high-speed centrifuge. Re-suspend the cell pellets in 12 mL of homogenization buffer (HB, Table 1) by pipetting.
		<ol>
			<li>Transfer the suspension to a single 50 mL plastic tube. Store the samples at -80 &#176;C or proceed to step 1.1.3.</li>
		</ol>
		</li>
		<li>Pre-cool the bead mill and equipment. Fill the 50 mL beaker of the bead mill to 75% with a glass bead mixture and add the cell suspension. For cell disruption, use 7 x 45 s pulses at full speed with 30 s of cooling between each pulse. Take 20 &#181;L of disrupted cells for quality checks in step 1.1.6.</li>
		<li>Filter the disrupted cells over a glass filter funnel and wash by pouring the HB over the glass beads until they appear clear. Pool the wash fraction with the filtrate. Keep the final volume lower than 150 mL.</li>
		<li>Centrifuge the sample for 15 min at 140 x g using three 50 mL plastic tubes to pellet the cell debris. Carefully transfer the supernatant to 20 mL polycarbonate ultracentrifugation vials and discard the pellet.
		<ol>
			<li>Fill the vials with HB, equilibrate the weight, and centrifuge in a suitable rotor for 1 h at 300,000 x g and 4 &#176;C to pellet the thylakoid membranes.</li>
		</ol>
		</li>
		<li>Use the centrifugation time to check the proportion of disrupted cells by microscopy at 400X magnification with the 20 &#181;L sample taken in 1.1.3. Calculate the ratio between chloroplast free and chloroplast containing frustules (silica shells). 
		Note: Diatom cell walls are made of silica, which is visible as highly diffracting substance in the microscope. Chloroplasts occurring as green dots should have been released from the cells if the cell disruption worked.</li>
		<li>Re-suspend the membrane pellet with as little washing buffer as possible (0.5-1 mL) using a small painter&#8217;s brush. Fill the polycarbonate ultracentrifugation vials with washing buffer (Table 1), equilibrate their weight and centrifuge for 20 min at 200,000 x g and 4 &#176;C.
		<ol>
			<li>Re-suspend the washed membranes with a painter&#8217;s brush. Add washing buffer only if needed to keep the thylakoid concentration as high as possible. Pool all thylakoids in one sample vial (15 mL).</li>
		</ol>
		</li>
		<li>Pre-dilute the samples using 10 &#181;L of the sample with 90 &#181;L of 100% acetone. Centrifuge it at 12,000 x g for 5 min to pellet precipitated protein. Take 10 &#181;L of the predilution and mix it with 990 &#181;L of 90% acetone.
		<ol>
			<li>Measure the absorbance (ABS) of chlorophyll a and c at 664 nm and 630 nm in 90% acetone. Subtract the ABS750nm from both values. Determine the total chlorophyll content using the following formula:24 
			1)&#160;&#160;&#160;&#160;&#160;&#160;&#160;<img alt="Equation 1" src="/files/ftp_upload/58017/58017eq1.jpg" /> 
			2)&#160;&#160;&#160;&#160;&#160;&#160;&#160;<img alt="Equation 2" src="/files/ftp_upload/58017/58017eq2.jpg" /></li>
		</ol>
		</li>
		<li>Aliquot thylakoids in portions of 0.5 mg of total chlorophyll in a 1.5 mL reaction tubes, freeze them in liquid nitrogen and store them at -80 &#176;C until further use.</li>
	</ol>
	</li>
	<li>Separation and concentration of FCP complexes
	<ol>
		<li>Prepare a sucrose gradient solution and fill the ultracentrifuge tubes until the top minus the loading volume (300 &#8211; 500 &#181;L). Freeze the tubes at -20 &#176;C until they are completely frozen. Allow the tubes to thaw at +4 &#176;C, which takes 3-4 h for a 17 mL tube.
		<ol>
			<li>Repeat the freeze-thaw cycle twice to refine the gradient for better resolution.</li>
		</ol>
		</li>
		<li>Use the samples obtained in 1.1.9 corresponding to 0.5 mg of chlorophyll and adjust with buffer B1 (Table 1) to a final volume of 2 mL. For solubilization, add n-Dodecyl-&#946;-D-maltopyranoside (b-DDM) to a final concentration of 20 mM.
		<ol>
			<li>Invert the tube 3 times and place it on ice for 20 min with gentle shaking to avoid foam. Centrifuge for 5 min, at 12,000 x g in a pre-cooled table top centrifuge at +4 &#176;C.</li>
		</ol>
		</li>
		<li>Load the supernatant on the gradient. Do not load more than 125 &#181;g of total chlorophyll per gradient if 17 mL vials are used. Centrifuge for 22 h at 100,000 x g and +4 &#176;C.
		<ol>
			<li>Recover the desired brown FCP fractions from the gradient using a syringe (Figure 2A). Take a 5 &#181;L aliquot and dilute it with 995 &#181;L of B1a.</li>
		</ol>
		</li>
		<li>Measure the absorbance (ABS) spectrum between 370 - 750 nm in a UV-VIS-spectrophotometer. Use semi-micro optical glass cuvettes.
		<ol>
			<li>Open the spectra manager software, go to spectrum mode and record a baseline from 370 to 750 nm followed by sample measurements. Use the following settings: scan speed, 200 nm/min; data pitch, 0.5 nm; response, medium.</li>
		</ol>
		</li>
		<li>Measure the volume of the recovered sample with a micropipette. Wash the FCP complexes by adding twice the recovered volume with B1 buffer (Table 1). Concentrate in a membrane concentrator with a 30 kDa cutoff at 1,000 x g and +4 &#176;C to an ABS672nm of at least 20. 
		Note: The b-DDM concentration might rise due to micelle enrichment in the sample compartment. This could lead to further solubilization of the FCP complexes! Avoid over-solubilization by adding detergent free buffer B1 if further washing steps are required to remove residual sucrose.</li>
		<li>Take a 20 &#181;L aliquot for the controls. Shock freeze the samples in liquid nitrogen and store them at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58017/58017fig2.jpg" /> 
Figure 2: Purification of FCP, spectroscopic controls and purity check. (A) Typical appearance of a sucrose density gradient after overnight centrifugation. All brown bands contain the FCP pool consisting of FCPa and FCPb. pigm.- unbound pigments, PS - photosystems (B) Absorbance spectra of FCP before (blue line) and after (orange-dashed line) concentration using centrifugal filter devices with 30 kDa cutoff. Particularly, carotenoids are prone to loss from the FCP, which would result in lower absorbance in the region between 500-550 nm. Graphs are normalized to the chlorophyll Qy maximum at ~670 nm. (C) Chlorophyll a emission spectra with excitation of chl c (465 nm) for testing the functional excitation energy transfer. If the energy transfer of chl c to chl a is hampered, an additional fluorescence band at ~640 nm (chl c) would occur. Graphs are normalized to the emission maximum. (D) Excitation spectra recorded at 675 nm (chl a fluorescence maximum) for testing the energy transfer to chl a from all pigments absorbing between 370 nm and 600 nm. If the energy transfer to chl a is less efficient, the fluorescence yield would decrease especially between 465 and 550 nm. The graphs are normalized to the maximum around 440 nm. The spectra in (B), (C) and (D) are nearly identical if the concentration worked well. (E) Check for purity of the isolated FCP using a Tris-tricine gel28. FCPa and FCPb have subunits between 18-19 kDa. All visible silver-stained proteins larger than 20 kDa are contaminants. Thyl. - Thylakoids <a href="https://www.jove.com/files/ftp_upload/58017/58017fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Spectroscopic and gel-based controls
	<ol>
		<li>Record the absorbance between 370-750 nm of 5 &#181;L of FCP in 995 &#181;L of B1a after step 1.2.5. Compare it with the spectrum obtained in step 1.2.4. Use the same instrumentation and settings as described in step 1.2.4.
		<ol>
			<li>Export data as *.csv and import the data into a spreadsheet. Normalize both spectra to the maximum of chl a in the Qy band of around 672 nm as depicted in Figure 2B.</li>
		</ol>
		</li>
		<li>Calculate the dilution factor of the samples from step 1.2.4 and 1.3.1 by dividing the measured ABS672nm by the desired ABS672nm of 0.03. Dilute the samples accordingly with B1a and transfer them to special glass fluorescence cuvettes.</li>
		<li>Record chl a fluorescence emission spectrum with a spectrofluorometer to reveal intact light energy transfer from chl c to chl a (Figure 2C).
		<ol>
			<li>Use the spectrometer software and go to the spectrum measurement mode with the following settings: mode, emission; slit width excitation and emission, 3 nm; sensitivity, medium; scan speed, 100 nm/min; data pitch, 0.5 nm; excitation wavelength, 465 nm; emission, 600-800 nm. Perform autozero and measure.</li>
		</ol>
		</li>
		<li>Record excitation spectra with the same sample and equipment to reveal intact energy transfer from all pigments to chl a (Figure 2D). Change settings to: mode, excitation; emission wavelength, 675 nm; excitation, 370-600 nm. Record the spectra.
		<ol>
			<li>Correct with a rhodamine spectrum in the same range for spectral properties of the lamp &#8211; cf. instructions in the user manual.</li>
		</ol>
		</li>
		<li>Mix FCP samples corresponding to 1 &#181;g of chl a with 10 &#181;L of SDS-loading buffer. Incubate for 10 min at 25 &#176;C. Centrifuge for 5 min at 12000 x g in a table top centrifuge.
		<ol>
			<li>Load the supernatant on a Tris-tricine gel28. Separate it for 2 h at 150 V and silver-stain it after separation40. 
			Note: The FCP subunits separate into two prominent protein bands between 18-19 kDa, which are constituents of FCPa and FCPb29 (Figure 2E).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Preparation of Liposomes and Incorporation of FCP
<ol>
	<li>Preparation of the lipid mixture and lipid detergent micelles 
	Note: Lipids are susceptible to warm temperatures combined with oxidative conditions. Try to keep the lipids chilled and under an N2 atmosphere.
	<ol>
		<li>Calculate the desired thylakoid lipid ratios for C. meneghiniana according to Vieler et al. 200730. Refer to the example given in Supplemental Table 1. Prepare the lipid stock solutions recommended by the manufacturer in a solvent proof container.</li>
		<li>Pipette the desired amount of lipids in a 2 mL reaction tube and evaporate the chloroform using a gentle nitrogen flow and try to spread the lipids over the whole area of the tube base. Let the N2 flow until all solvent is evaporated.</li>
		<li>Solubilize the lipid mixture in 29 &#181;L of n-octyl &#946;-D-glucopyranoside solution (OG) at 4 &#176;C for 4 h. Incubate the lipid mixture for 10 min at 30 &#176;C. Incubate the lipids in a sonicator bath for 3 x 3 min at 25 &#176;C interrupted by 30 s on ice.
		<ol>
			<li>Add 221 &#181;L of tricine buffer and 250 &#181;L of 4x dialysis buffer.</li>
		</ol>
		</li>
		<li>Use an extruder with 0.1 &#181;m polycarbonate membranes for a defined liposome diameter of 50-70 nm. Assemble the extruder with the membrane and filter support. Avoid air bubbles and tighten the assembly thoroughly.</li>
		<li>Fill one syringe with 4x dialysis buffer and pre-wet the extruder until no bubbles can be seen in the second syringe.</li>
		<li>Apply the lipid-detergent-micelles to the extruder and press the solution from one syringe to the other forth and back. Repeat this step 5 times until the solution appears homogenous. 
		Note: This solution can be stored at 4 &#176;C for several days. Do not freeze!</li>
	</ol>
	</li>
	<li>Incorporation of FCP complexes and removal of detergents and aggregates 
	Note: In this example we use a lipid/chl a ratio of 12:1, which corresponds to a lipid/protein ratio of roughly 100:1.
	<ol>
		<li>Add FCP equal to 20 &#181;g of chl a in a total volume of 500 &#181;L of B1a buffer to 250 &#956;L of the extruded lipid-micelles and 250 &#181;L of 4x dialysis buffer. Incubate the samples for 3 x 3 min at 25 &#176;C in a thermomixer at 1,500 - 3,000 rpm interrupted by a 30 s pause on ice.</li>
		<li>Cut the lid of four 1.5 mL reaction tube just under the top giving a ring which still fits on the lid. Prepare 1.5 cm x 1.5 cm pieces of dialysis membrane and wash them in 20 mL of 1x dialysis buffer</li>
		<li>Fill 250 &#181;L of the sample to each lid. Carefully, lay the membrane on the lid so that the compartment is completely filled with the sample and no air bubbles occur. Tighten the reaction tube ring on the assembly in order to have a closed compartment.</li>
		<li>Dialyze the samples in 50 mL of 1x dialysis buffer overnight (12-16 h) on ice on a tumbling shaker. Replace the used dialysis buffer with fresh one and add 7 mg of adsorbent beads to remove the remaining detergents for at least 6 h.</li>
		<li>Replace the dialysis buffer again and dialyze for another 12 h. Recover the liposomes by piercing the dialysis membrane with a 200 &#181;L micropipette tip and aspirate all liposomes from the reaction tube lid.</li>
		<li>Optional step: If high purity (&#62;95%) is needed. Prepare a discontinuous density gradient in 17 mL ultracentrifugation vials with steps containing 6%, 10%, 15% and 20% sucrose epichlorhydrin copolymer in dialysis buffer. Load the liposomes on top and ultracentrifuge at 100,000 x g for 4 h in a swinging bucket rotor.
		<ol>
			<li>Recover the upper brown band with a syringe, dilute the sample 1:5 with DP and proceed to the next step.</li>
		</ol>
		</li>
		<li>Centrifuge the FCP liposomes in at least 2 mL of 1x dialysis buffer for 1.5 h at 100,000 x g and 4 &#176;C. Recover the liposomes by turning the centrifuge tube at an angle of 45&#176;. Allow the liposomes to move down for 1 min (Figure 3A).</li>
		<li>Recover the FCP liposomes in a final volume of 25-50 &#181;L. Avoid disturbing the precipitate.</li>
	</ol>
	</li>
	<li>Controls 1: absorbance, fluorescence spectroscopy
	<ol>
		<li>Add a 3 &#181;L aliquot of the FCP-liposomes to a 1 mL final volume of 1x dialysis buffer and centrifuge for 5 min at 12,000 x g.
		<ol>
			<li>Record the absorbance between 370 and 750 nm of FCP-liposomes with the same equipment as described in 1.2.4. Normalize the spectrum to the maximum in the Qy region of chl a (670-680 nm) and compare it to the normalized spectrum of solubilized FCP (Figure 3C).</li>
		</ol>
		</li>
		<li>Prepare FCP-liposomes in 1 mL of DP with an absorbance (ABS)=0.03 with respect to the maximum between 670-680 nm. Adjust the solubilized FCP in detergent from step 1.2.6 to the same ABS diluting with B1a.</li>
		<li>Record absorbance spectra of both samples as described in 1.2.4. Record chl a fluorescence emission spectrum of both samples as described in 1.3.3. 
		Note: The fluorescence yield is decreased in the FCP-liposome sample (Figure 3D and cf. discussion.)</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58017/58017fig3.jpg" /> 
Figure 3: Isolation of FCP proteoliposomes followed by spectroscopic controls and confocal imaging. (A) Recovery of FCP liposomes after centrifugation. Turn the centrifugation tube to 45&#176; and wait approximately 1 min - the liposomes will move down whereas the FCP aggregates which are not incorporated into liposomes stick to the tube wall. (B) Comparison of absorbance spectra of solubilized FCP in detergent (blue) and FCP in liposomes (orange) (C) The same spectra as in (B) normalized to chl a maximum in the red region (~670 nm - Qy peak); solubilized FCP in detergent (blue) and FCP in liposomes (orange). Potentially, there could be a pigment loss mainly of carotenoids visible in the 500-550 nm region. The clustering of FCP in the liposomes may lead to a peak broadening and a slight shift of the chl a maximum (~670 nm) to the red. (D) Emission spectra of solubilized FCP in detergent and FCP in liposome. Clustering of FCP in the liposome enhances energetic interactions of FCP complexes which lowers the fluorescence yield (orange curve) and shifts the emission maxima slightly to the red. <a href="https://www.jove.com/files/ftp_upload/58017/58017fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The authors have nothing to disclose.

FCP liposomes with natural lipid composition provide a handy, simple and reproducible tool to investigate spectroscopic properties in vitro. The lipid environment in FCP liposomes resembles the situation within the thylakoid membrane, giving rise to experimental results that are closer to natural conditions.
There are several advantages of using C. meneghiniana as a model system for FCP antenna. It grows relatively fast and is more robust in comparison to other diatom model s...

Photosynthetic organisms such as diatoms must cope with ever-changing light conditions and respond with sophisticated acclimation mechanisms that sustain high photosynthetic efficiency and protect from photo-oxidative damage caused by the excessive light. A major light-protective process in photosynthetic eukaryotes is the high energy quenching (qE) of absorbed light that occurs as the main contribution to the non-photochemical quenching (NPQ) under light stress conditions1,2,3. The light harvesting antenna complexes (LHC) are involved in the regulation of excitation energy transfer pathways. In response to high light induced low pH in the chloroplast lumen, the antenna system switches from the light harvesting state to the quenching state. This energy dissipative state protects photosystems (PS) and other complexes in the thylakoid membrane from photo-oxidation. In photosynthetic eukaryotes, the qE is usually induced by two factors1,2,3. One factor is the specialized light harvesting protein that responds to the low pH. The PsbS protein induces the qE in higher plants4. LhcSRs5, modulated by PsbS activity, induce the qE in green algae6. Diatoms possess Lhcx-like proteins which structurally related to LHCSRs7,8,9,10.
The second factor of qE is the xanthophyll cycle where carotenoids of the antenna are converted into a photo-protective form by de-epoxidation and reverted by epoxidation. In plants and green algae, violaxanthin is converted to zeaxanthin. In diatoms, diadinoxanthin is converted to diatoxanthin, which then correlates with the extent of NPQ11. The diatom light harvesting antenna possesses some peculiarities although it is evolutionary related to plant and algal LHCs. The switch from light harvesting to photo-protection is enormously fast and the NPQ capacity is higher compared to plants12. This might be one reason why diatoms are very successful in different ecological niches in a way that they are responsible for up to 45% of the oceanic net primary production13. Therefore, diatom light harvesting systems are an interesting object of photosynthesis research.
Diatoms, like the centric species Cyclotella meneghiniana, possess thylakoid intrinsic light harvesting systems named after the pigments they bind - fucoxanthin, chlorophyll (chl) a and c, hence FCP. Light harvesting proteins, such as FCPs, are embedded in the thylakoid membrane system comprising several membrane layers. Diatoms form bands of three thylakoids. This complex situation makes it difficult to study them on the molecular level in the thylakoid membrane. In addition, many components contribute to the regulation of light harvesting (see above). Therefore, in many approaches, the complexes were isolated from the membrane using mild detergents, such as n-Dodecyl-&#946;-D-maltopyranoside (&#946;-DDM), which solubilize the membrane but keep the FCP complexes intact. Many spectroscopic studies were performed using solubilized FCP to investigate intramolecular energy transfer14,15,16,17. However, this former approach was limited since the regulation of energy transfer needs excitonic interaction with other antenna complexes or photosystems. Hence, these kinds of studies cannot be carried out with solubilized complexes because the interaction between complexes is lost.
An important feature in antenna regulation is the &#34;molecular crowding&#34; of the antenna and photosystems in the thylakoid membrane18. Formerly, a simple approach was carried out to simulate this effect in vitro. The detergent was removed, which leads to random aggregation of antenna complexes. Although some reasonable data was obtained by this approach17,19, the detergent removal does not reflect the situation in vivo and has some limitations since the complexes are not interacting in their regular tertiary structure.
The use of liposomes overcomes several of the former limitations. The tertiary structure is still fully intact. The liposome membrane provides a quasi-native environment for the antenna complexes. The membrane separates the inside of the liposome from the outside environment. By these means, liposomes provide two reaction compartments for studies of ion and pH gradients as well as for transport processes. Further, the parameters of the experimental system can be controlled more easily for studies in the thylakoid membrane. Liposomes were already shown to be an excellent tool to study photosynthetic complexes. A major focus in the past was on plant LHC where the effect of altered lipid composition was tested on LHC II20. In other approaches, protein-protein interaction between different LHC II were investigated21. Also, some studies in green algae were carried out that describe spontaneous clustering between LHC22. Considering the importance of diatoms for aquatic ecosystems, relatively few studies were performed with antenna complexes of diatoms. Two studies investigated the antenna complexes of the centric Cyclotella meneghiniana, where the clustering of the FCP antenna23 and responsiveness of FCP to electrochemical gradients24 were shown. Thus, liposomes are an excellent tool to study diatom antennas and their interaction and regulation in nearly native conditions. The liposomes are versatile since many conditions such as lipid composition, liposome size, protein density and the surrounding aqueous phase can be controlled. Furthermore, the method requires low amounts of samples. The experimental system is robust and highly reproducible. The compartmentalization of liposomes allows for studying pH and ion gradients, which are important factors in the regulation of antenna complexes.
Here, we describe the isolation of FCP antenna complexes from C. meneghiniana and their incorporation in liposomes with natural thylakoid lipid composition. Also, we provide exemplary data for the spectroscopic characterization of solubilized FCP and compare them with FCP in liposomes. The method summarizes knowledge and standardized protocols obtained from the improvements of Gundermann and B&#252;chel 201223, Natali et al. 201622, and Ahmad and Dietzel 201724.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58017/58017fig1.jpg" /> 
Figure 1: Schematic representation of the workflow. (1) Refers to paragraph 1 which describes cell growth, disruption and thylakoid isolation with following FCP separation on sucrose density gradients; C. m.-Cyclotella meneghiniana cells. (2) Preparation of natural thylakoid lipid mixture (MGDG, DGDG and SQDG) described in paragraph 2 and creation of lipid-detergent micelles with octylglycoside (OG). A defined lipid-micelle size is achieved by extrusion using membranes of a defined pore diameter. FCP and lipid-micelles are unified at a predefined lipid: protein ratio and the detergents OG and &#946;-DDM are removed via controlled dialysis forming FCP proteoliposomes. <a href="https://www.jove.com/files/ftp_upload/58017/58017fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>500 ml centrifuge vials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>high speed centrifuge</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bead Mill VI 2</td>
			<td>Edmund-B&#252;hler (edmund-buehler.de)</td>
			<td></td>
			<td>newer version: Vibrogen-Zellm&#252;hle Vl 6</td>
		</tr>
		<tr>
			<td>Silibeads S 400 &#181;m</td>
			<td>Sigmund-Lindner.com</td>
			<td>5223-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Silibeads S 1,-1,3 mm</td>
			<td>Sigmund-Lindner.com</td>
			<td>4504</td>
			<td></td>
		</tr>
		<tr>
			<td>VitraPOR filter funnel - por1</td>
			<td>ROBU GmbH</td>
			<td>21121</td>
			<td></td>
		</tr>
		<tr>
			<td>polycarbonate ultracentrifuagtion vials (30 mL) for T-865</td>
			<td>Beranek Laborger&#228;te (Laborgeraete-beranek.de)</td>
			<td>314348</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge Discovery 90SE</td>
			<td>Sorvall</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>rotor T 865</td>
			<td>ThermoFisher Scientific (thermofisher.com)</td>
			<td>51411</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer Cell Counter Chamber (improved)</td>
			<td>Carl Roth Laborbedarf (Carlroth.com)</td>
			<td>T729.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Mikroskop Primostar (7)</td>
			<td>Optik-Pro (optik-pro.de)</td>
			<td>51428</td>
			<td></td>
		</tr>
		<tr>
			<td>optical glass cuvettes (6040-OG)</td>
			<td>Hellma Analytics (hellma-analytics.com)</td>
			<td>&#34;6040-10-10&#34;</td>
			<td></td>
		</tr>
		<tr>
			<td>V-630 UV-VIS Spectrophotometer (incl. software)</td>
			<td>Jasco (jasco.de)</td>
			<td>V-630</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Dodecyl-&#946;-D-Maltopyranoside</td>
			<td>ANATRACE (anatrace.com)</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Clear tubes 17 ml for AH629</td>
			<td>Beranek Laborger&#228;te (Laborgeraete-beranek.de)</td>
			<td>344061</td>
			<td></td>
		</tr>
		<tr>
			<td>rotor AH629-17-mL</td>
			<td>ThermoFisher Scientific (thermofisher.com)</td>
			<td>54285</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane concentrator_Centriprep 30 kDa cutoff</td>
			<td>Millipore (merckmillipore.com)</td>
			<td>4307</td>
			<td></td>
		</tr>
		<tr>
			<td>Biometra Minigel-Twin</td>
			<td>Analytik Jena AG (analytik-jena.de)</td>
			<td>846-010-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Stain Plus Kit</td>
			<td>Bio-Rad (bio-rad.com)</td>
			<td>1610449</td>
			<td></td>
		</tr>
		<tr>
			<td>libre office spread sheet</td>
			<td>The document foundation</td>
			<td>https://de.libreoffice.org/download/libreoffice-still/</td>
			<td></td>
		</tr>
		<tr>
			<td>special glass cuvettes for fluorescence (101-0S)</td>
			<td>Hellma Analytics (hellma-analytics.com)</td>
			<td>101-10-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrofluorometer FP-6500 (incl. Software)</td>
			<td>Jasco (jasco.de)</td>
			<td>FP-6500</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-loading buffer Roti-Load</td>
			<td>ROTH (carlroth.com)</td>
			<td>K929.1</td>
			<td></td>
		</tr>
		<tr>
			<td>n-octyl &#946;-D-glucopyranoside</td>
			<td>ANATRACE (anatrace.com)</td>
			<td>O311</td>
			<td></td>
		</tr>
		<tr>
			<td>Monogalactosyl Diaclyglycerol (MGDG)</td>
			<td>Larodan AB (larodan.com)</td>
			<td>59-1300</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>Digalactosyl Diacylglycerol (DGDG)</td>
			<td>Larodan AB (larodan.com)</td>
			<td>59-1310</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>Sulphoquinovosyl Diacylglycerol (SQDG)</td>
			<td>Larodan AB (larodan.com)</td>
			<td>59-1230</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>L-alpha-Phosphatidylglycerol (PG)</td>
			<td>Larodan AB (larodan.com)</td>
			<td>37-0150</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>L-&#945;-Phosphatidylcholine</td>
			<td>Sigma-Aldrich (sigmaaldrich.com)</td>
			<td>P3782 SIGMA</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>sonicator bath S-50TH</td>
			<td>Sonicor (getmedonline.com</td>
			<td>SONICOR-S-50TH</td>
			<td></td>
		</tr>
		<tr>
			<td>mini-Extruder</td>
			<td>Avanti Polar Lipids (Avanti.com)</td>
			<td>610000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuleopore polycarbonate membrane</td>
			<td>Avanti Polar Lipids (Avanti.com)</td>
			<td>610005</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis membrane Visking 14 kDa cutoff</td>
			<td>ROTH (carlroth.com)</td>
			<td>0653.1</td>
			<td>boil in destilled water before use</td>
		</tr>
		<tr>
			<td>Biobeads SM2 Adsorbent</td>
			<td>Biorad (Bio-rad.com)</td>
			<td>152-3920</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose epichlorhydrin copolymer - Ficoll 400</td>
			<td>Sigma-Aldrich (sigmaaldrich.com)</td>
			<td>F4375</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate ultracentrifuagtion vials (2.7 mL) for TFT 80.4</td>
			<td>Beranek Laborger&#228;te (Laborgeraete-beranek.de)</td>
			<td>252150</td>
			<td></td>
		</tr>
		<tr>
			<td>rotor TFT 80.4</td>
			<td>Millipore (merckmillipore.com)</td>
			<td>54356</td>
			<td></td>
		</tr>
		<tr>
			<td>material listed in order of appearance </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>For specific safety instructions please refer to material safety sheets and repective manuals.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard lab material and substances are not listed.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating and incorporating light-harvesting antennas from diatom cyclotella meneghiniana in liposomes with thylakoid lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

The protocol describes the isolation of total FCP fraction from Cyclotella meneghiniana and incorporation into liposomes with native lipid composition. The thylakoid isolation is highly reproducible, but the thylakoid yield may change. The result is acceptable if more than 50% of all pigments are recovered in step 1.1.4. More than 80% is optimal.
The solubilization of the thylakoids is a critical step. Well-solubilized ...

Watch this Scientific Journal Video about Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids at JoVE.com

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

isolating-incorporating-light-harvesting-antennas-from-diatom

Universidad San Sebastian

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JoVE Journal

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Cell Biology

Biochemistry

Unit 1

Chloroplasts and Photosynthesis

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7.7K Views.  Goethe University. Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

Video: Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a &#39;CAAX&#39; box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.

Prenylation is an important modification on peripheral membrane binding proteins. Insect cells can be manipulated to produce farnesylated and carboxymethylated KRAS4b in quantities that enable biophysical measurements of protein-protein and protein-lipid interactions

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The &#8220;tubulin code&#8221; is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs.
To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps.
We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.

This protocol describes tubulin purification from small/medium-scale sources such as cultured cells or single mouse brains, using polymerization and depolymerization cycles. The purified tubulin is enriched in specific isotypes or has specific posttranslational modifications and can be used in in vitro reconstitution assays to study microtubule dynamics and interactions.

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They ...