We acknowledge funding from the United Kingdom&#39;s Department of Business, Energy, and Industrial Strategy (BEIS). We thank Alex R. Jones and Mike Shaw from the National Physical Laboratory for their feedback on the manuscript.

1. Protein sample preparation
<ol>
	<li>Prepare (through recombinant methods or otherwise) and purify the protein(s)&#160;of interest in a suitable buffer (i.e., one that is amenable for protein stability and crystallization) that has been filtered with a 0.22 &#181;m filter (see Table of Materials). Evaluate the protein quality (in terms of purity, polydispersity, and stability) prior to crystallization31. 
	NOTE: Please see the representative results section for details on the proteins and buffers used in the present study.</li>
	<li>Concentrate the sample up to 10-50 mg.mL-1 or higher (depending on the protein target) using the correct MWCO concentrator (see Table of Materials). 
	NOTE: The concentrated sample can be used immediately for crystallization or stored frozen at -80 &#176;C.</li>
	<li>If the protein has been frozen, centrifuge the sample for 10 min in a bench-top centrifuge (&#8805;16,000 &#215; g, at room temperature or 4 &#176;C) to pellet and remove any unwanted material (e.g., precipitates).</li>
	<li>Aliquot the protein into clean 200 &#181;L PCR tubes. Keep it on ice or at 4 &#176;C if the protein is unstable at room temperature.</li>
</ol>
2. Setting up the microdialysis plate
NOTE: The commercially available microdialysis plate (see Table of Materials) consists of two sides (&#39;protein side&#39; and &#39;buffer side&#39;) with a regenerated cellulose membrane (available in different MWCOs), as shown in Figure 2.
<ol>
	<li>With the plate orientated protein side up, peel back and remove the adhesive cover tape (Figure 2A) from the 200 &#181;m pressure adhesive spacer. Take note of the position of well A1.</li>
	<li>Using a multichannel pipette, load a maximum of 3.2 &#181;L of protein (step 1.4) into each well (Figure 2B). 
	NOTE: The protein can also be loaded using a repeat dispensing pipette. Using a single-channel pipette is possible, but it will increase the likelihood of partial dehydration of the drops occurring due to the extended loading time.</li>
	<li>Position the 200 &#181;m UV cover film (see Table of Materials) over the 96-well plate and check its integrity (Figure 2C). Ensure that the protective film is facing up.</li>
	<li>Use a sealing paddle (see Table of Materials), press down the UV cover film to activate and seal the pressure adhesive.</li>
	<li>Invert the plate and take note of the position of well A1. Ensure that the plate is oriented buffer side up and that the well positions are mirrored (Figure 2D).</li>
	<li>Using a multichannel pipette, load a maximum of 350 &#181;L of the dialysis solution into each of the wells (Figure 2D). 
	NOTE: In this step, either commercially available crystallization screens (sparse matrix screens) or laboratory-made customized screens (grid screens) can be used (see Table of Materials).</li>
	<li>Carefully seal the wells with the &#39;reservoir cover film&#39; (see Table of Materials) (Figure 2E).</li>
	<li>Place the plate (buffer side up) in a suitable temperature-controlled incubator (20 &#176;C) for crystal growth. 
	NOTE: For the proteins selected for the present study, the crystals started to appear between 1-8 h at 20 &#176;C.</li>
	<li>To inspect for crystals under the microscope, invert the plate to bring the protein side up and remove the protective film from the 200 &#181;m UV cover film (step 2.3), as shown in Figure 2F,G.</li>
</ol>
3. Large-scale crystallization using dialyzer tubes
NOTE: Once crystallization conditions have been explored using the microdialysis plate, large-scale crystallization of the protein (for serial crystallography or other purposes) can be performed using the dialyzer tube, as shown in Figure 3. Similar to the microdialysis plate, these devices are available with 3, 5, 10, and 30 kDa MWCO dialysis membranes.
<ol>
	<li>Prepare the optimized crystallization condition for the growth of microcrystals in large containers (adopting the conditions optimized using the microdialysis plate). Ensure the buffer has been previously filtered with a 0.2 &#181;m filter.</li>
	<li>Pipette the protein into the dialyzer tube (maximum volume of 0.5 mL) and seal the end using the included red plastic cap (Figure 3A).</li>
	<li>Place the 500 &#181;L dialyzer tube into a suitably sized container (depending on the total volume of dialysis solution) containing the floating rack (see Table of Materials), as shown in Figure 3B. 
	NOTE: The floating rack provided with the dialyzer kit holds up to 18 dialyzer tubes simultaneously.</li>
	<li>Place the container stationary in a suitable temperature-controlled incubator (20 &#176;C) for crystal growth.</li>
	<li>To inspect for crystals, pipette 1-2 &#181;L of the solution onto a glass cover slide. Cover with a second glass cover slide on top (to prevent excess dehydration) and view under a microscope (see Table of Materials). 
	NOTE: Crystals may be damaged when sandwiched between two glass cover slides during the inspection. Crystals may also be viewed directly by placing the dialyzer tube under a stereo high-magnification microscope.</li>
</ol>

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The co-authors Pascelle Draper and Paul Reardon are employed by SWISSCI.

Currently, crystallization by dialysis is the most underutilized crystallization method, namely due to the low-throughput and tedious nature of the existing approaches, such as microdialysis with buttons. Here, a simple but robust protocol follows an HTP workflow for protein crystal growth by dialysis through a commercially available microdialysis plate and dialyzer tube. Depending on the target protein, the microdialysis plate and dialyzer tube used in the procedure come with a choice of a dialysis membrane with a 3, 5, 10, or 30 kDa MWCO. The protocol can be easily set up in any standard crystallization facility and has the great advantage of being applicable to both soluble and membrane proteins. However, protein-protein and protein-nucleic acid complexes were not tested during this protocol.
As in any crystallization approach by dialysis, the volume ratio between the protein sample and dialysis solution is critical. In this protocol, a ratio of 1:100 between the sample and dialysis solution is recommended, but since the microdialysis plate allows a maximum capacity of 350 &#181;L of dialysis solution, these ratios can be explored to obtain crystal hits. A volume of 1-2 &#181;L of protein is used in the presented protocol when setting up crystallization plates. This is to ensure that drops set accurately with a manual multichannel pipette. By using electronic pipettes (multichannel or repeat dispensing pipettes) or HTP liquid dispensing robots, drops of lower volumes can be achieved accurately, thus lowering the amount of protein required. Furthermore, due to the relatively low volumes of dialysis buffer required by the microdialysis plate (contrary to other conventional dialysis methods), it is possible (without the extensive use of resources) to explore large chemical spaces, not only using commercially available crystallization screens but also with optimization screens (designed around the initial crystallization hit condition).
A critical step in the presented HTP procedure is the timely application of small protein volumes (0.50-3.2 &#181;L per condition) to the dialysis plate to limit dehydration and sample loss. This can be easily mitigated by using a multichannel pipette, a repeat dispensing pipette, or a robotic crystallization system. The long incubation time, such as more than 2 weeks, of the plates at 20 &#176;C might lead to dehydration of the protein droplets or damage to the recently formed crystals. Keeping the dialysis plates inside a humidification chamber or a sealable bag can alleviate this effect. In addition, using sterile materials and techniques are recommended to avoid bacterial growth.
As mentioned in the introduction, recently, with the increased need to understand protein structural dynamics for disease mechanisms, protein-ligand binding interactions, and protein-protein interactions, the field of protein X-ray crystallography has been revolutionized through the development of new and existing crystallization techniques, modern approaches to crystal sample delivery, new generations of X-ray sources, and new sophisticated methods for data acquisition and processing36,37,38. Therefore, the advent of room temperature serial micro-crystallography, either performed using XFELs or synchrotron light sources, has emerged as a remarkable tool in structural biology, specifically in the field of membrane proteins39. However, thousands of microcrystals are required to generate enough data for a robust structure solution, which is not an easy task (at least by conventional crystallization methods). The dialysis crystallization method described here enables the production of a large number of microcrystals. Once the crystallization condition for the production of microcrystals (1-10 &#181;m) has been determined through the use of a microdialysis plate, large quantities of high-density microcrystals can be produced using the 0.5 mL dialyzer device (Figure 5).&#160;These crystals are ideally suited for data collection using fixed targets or liquid jet sample delivery systems27,40. Crystals obtained through this method can also be appropriate for MicroED applications. However, these may need to be milled down to a suitable size and thickness for this specific application, as electrons interact much more strongly with crystals than X-ray photons41.
In conclusion, the crystallization by dialysis approach described here adds to the evolving strategies in protein crystallization for structure determination and expands the range of efforts than can be employed to determine novel protein targets that have previously been unsuccessful with other conventional methods.

Over the last century, X-ray crystallography has been critical in elucidating and understanding the structure-function paradigm of biological macromolecules. To date, it continues to be one of the most successful methods in elucidating atomic resolution structures of many uniquely different proteins that are crucial to the fundamental understanding of cell biochemistry, medicine, and early drug discovery1,2. However, protein crystallization remains a bottleneck in studying many protein targets, particularly membrane proteins and large protein complexes3. Consequently, protein crystallization is almost always considered an art due to the labor-intensive trial-and-error approaches employed4,5,6.
A precipitating agent is usually added to a protein solution at high concentration to form a well-ordered, regular, and repeating lattice arrangement of protein molecules, known as crystals. Under favorable conditions, such as temperature, pH, concentration, and precipitant agent, a supersaturated solution eventually forms, followed by crystal nucleation and growth7,8. Although there have been many advances in crystallization trial setups, predominantly with the development of high-throughput robotic systems and the availability of ready-made &#34;sparse matrix&#34; screens, the general approaches to protein crystallization have largely remained unchanged over the years. Common experimental protein crystallization techniques include vapor diffusion (hanging drop and sitting drop)9, microbatch (under oil)10,11, free-interface diffusion (microfluidic devices)12, and dialysis (using buttons and other techniques)13,14,15. However, other more specialized setups also exist, such as mesophase approaches for crystallizing membrane proteins16,17. While the majority of X-ray protein structures deposited in the Protein Data Bank have so far been solved through crystallization by vapor diffusion methods6,18,other approaches, such as crystallization by dialysis, seem to be underutilized, likely due to the practical aspects related to their experimental setup.
Crystallization by dialysis simply relies on the slow diffusion of solutes (precipitants, ions, additives, and buffers) through a semi-permeable membrane that simultaneously prevents protein molecules from circulating. This way, the protein solution is slowly brought into equilibrium, with the precipitant reaching the necessary concentration to crystallize. The system&#39;s kinetics depends on temperature, precipitant concentration, and the cellulose membrane molecular weight cut-off (MWCO)19. To date, the most popular crystallization setup by dialysis has been using microdialysis buttons made of transparent acrylic sheets. These are usually immersed in reservoirs (mostly using vapor diffusion hanging drop plates) containing the crystallization precipitant solutions. However, this lower-throughput method also requires specific assembly to seal the protein solution within the dialysis membrane placed over the button chamber, as illustrated in Figure 1. Moreover, air bubbles trapped between the dialysis membrane and the protein solution are a frequent problem that impair crystal growth. Another constraint of the method is the sample requirements, whereby much higher concentrations and volumes are necessary compared to vapor diffusion methods, to accommodate the dialysis buttons. Therefore, crystallization using microdialysis buttons has been perceived as an unappealing method, especially for difficult targets such as membrane proteins, whose purification yields are frustratingly low. Recently, microfluidic devices have been developed to facilitate protein crystallization by dialysis15. These chips have also been designed to have high X-ray transparency with low background, allowing the chips to be used for in situ data collection at room temperature, thus eliminating the inconvenience of harvesting and cryocooling crystals. Despite these advances, the approach is still very low-throughput and expensive.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64744/64744fig01.jpg" /> 
Figure 1: Schematic representation of crystallization by dialysis using dialysis buttons. (A)&#160;Schematic representation of a crystallization dialysis button. (B) The protein solution is added to the microdialysis button chamber. (C) The dialysis membrane is held to the microdialysis button with the help of a rubber ring (O-ring) applied via an applicator. (D) The dialysis button is ready to be immersed in the reservoir containing the crystallization solution (dialysis solution), as shown in (E). The vial containing the immersed dialysis button must be sealed to avoid evaporation. <a href="https://www.jove.com/files/ftp_upload/64744/64744fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here, a straightforward protocol is presented for screening protein crystallization conditions and crystal growth using the 96-well high-throughput dialysis plate. These disposable plates are designed to be used similarly to the vapor diffusion crystallization plates (pipette then seal), as shown in Figure 2. The plates can accommodate up to 3.2 &#181;L of protein and 350 &#181;L of dialysis solution. Each well features a separate regenerated cellulose membrane to prevent cross-contamination between the wells. The setup takes around 10 min to complete and does not require any specialized equipment besides what can be found in all standard crystallization laboratories. Four different proteins, including two membrane proteins, are used to demonstrate and validate this approach as an effective method for high-throughput (HTP) protein crystallography.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64744/64744fig02.jpg" /> 
Figure 2: Crystallization workflow using the microdialysis plate. (A) Removal of the red adhesive &#34;cover film&#34;. (B) Dispensing the protein droplets into each of the drop wells. (C) The wells are covered with the UV &#34;cover film&#34;. (D) The plate is inverted to add the dialysis solutions (or crystallization screen). (E) The plate is sealed and incubated. (F,G) Microscope inspection of the drops. <a href="https://www.jove.com/files/ftp_upload/64744/64744fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The use of this crystallization by dialysis protocol was demonstrated using the 0.5 mL dialyzer tube (Figure 3) for the large-scale (hundreds to thousands) production of microcrystals, suitable for state-of-the-art data collection methods such as serial crystallography at both XFEL facilities20,21,22,23,24 and synchrotrons25,26,27, as well as for MicroED28,29,30 approaches.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64744/64744fig03.jpg" /> 
Figure 3: Large-scale microdialysis crystallization using the dialyzer tube. (A) Schematic representation of the 0.5 mL dialyzer tube. (B) Side view of a beaker containing the crystallization solution and the floating tube rack holding a dialyzer tube. <a href="https://www.jove.com/files/ftp_upload/64744/64744fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL tubes</td>
			<td>Thermo Scientific</td>
			<td>AB0620</td>
			<td>For aliquoting protein solutions.</td>
		</tr>
		<tr>
			<td>0.2 &#181;m syringe filter</td>
			<td>Sartorius</td>
			<td>17823----------K</td>
			<td>Surfactant-free cellulose acetate filters. For filtering dialysis solutions.</td>
		</tr>
		<tr>
			<td>0.22 &#181;m membrane filters</td>
			<td>Millipore</td>
			<td>GSTF04700</td>
			<td>Membrane filters for filtering large volumes of buffers</td>
		</tr>
		<tr>
			<td>12-channel, variable 0.5 &#8211; 10 &#181;L Research plus pipette</td>
			<td>Eppendorf</td>
			<td>3125000028</td>
			<td>For dispensing protein drops onto the Diaplate.</td>
		</tr>
		<tr>
			<td>12-channel, variable 30 &#8211; 300 &#181;L Eppendorf Research plus pipette&#160;</td>
			<td>Eppendorf</td>
			<td>3125000060</td>
			<td>For dispensing dialysis solutions on the Diaplate reservoirs.</td>
		</tr>
		<tr>
			<td>20 mL syringe</td>
			<td>Fisherbrand</td>
			<td>15889152</td>
			<td>For use with syringe filters.</td>
		</tr>
		<tr>
			<td>96 well 2.2 mL deep-well plates</td>
			<td>Thermo Scientific</td>
			<td>AB0788</td>
			<td>Polypropylene deep-well storage plates; for preparing screens using the Hamilton Microlab STARlet.</td>
		</tr>
		<tr>
			<td>Centrifuge 5425</td>
			<td>Eppendorf</td>
			<td>5405000565</td>
			<td>With rotor FA-24x2 with a maximum g-force of 21,300 x g.</td>
		</tr>
		<tr>
			<td>Diacon dialyser</td>
			<td>SWISSCI</td>
			<td>W72010</td>
			<td>Dialyzer tubes with a regenerated cellulose membrane with a molecular weight cut-off of 10 kDa. Ideal for protein solutions of up to 0.5 mL.</td>
		</tr>
		<tr>
			<td>Diaplate 96-well plate</td>
			<td>SWISSCI</td>
			<td>W82010</td>
			<td>Microdialysis plate. The Diaplate consists of two sides with a regenerated cellulose membrane in-between with a molecular weight cut-off of 10 kDa.</td>
		</tr>
		<tr>
			<td>Falcon 50 mL High Clarity PP Centrifuge Tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td>For holding dialysis solutions.</td>
		</tr>
		<tr>
			<td>Floating rack</td>
			<td>SWISSCI</td>
			<td>n/a</td>
			<td>Included in the Diacon kit</td>
		</tr>
		<tr>
			<td>Floor-standing vibration-free incubator</td>
			<td>Molecular Dimensions</td>
			<td>MD5-01</td>
			<td>400 L temperature-controlled incubator set to 20 &#176;C.</td>
		</tr>
		<tr>
			<td>Leica M205 C stereo microscope</td>
			<td>Leica</td>
			<td></td>
			<td>Planapo 1.0x objective, 7.8x &#8211; 160x zoom range with DMC 4500 camera</td>
		</tr>
		<tr>
			<td>Lysozyme from chicken egg white</td>
			<td>Sigma Aldrich</td>
			<td>62971</td>
			<td>Lyophilized protein</td>
		</tr>
		<tr>
			<td>Memgold2</td>
			<td>Molecular Dimensions</td>
			<td>MD1-64</td>
			<td>Sparse-matrix screen</td>
		</tr>
		<tr>
			<td>Microlab STARlet</td>
			<td>Hamilton</td>
			<td>n/a</td>
			<td>Liquid handler system.</td>
		</tr>
		<tr>
			<td>Reservoir cover film</td>
			<td>SWISSCI</td>
			<td>n/a</td>
			<td>Included in the Diaplate kit</td>
		</tr>
		<tr>
			<td>Reusable bottle top filter</td>
			<td>Thermo Scientific</td>
			<td>DS0320-5045</td>
			<td>For fitering large volumes of buffers, for use with 0.22 &#181;m membrane filters</td>
		</tr>
		<tr>
			<td>Sealing paddle</td>
			<td>SWISSCI</td>
			<td>n/a</td>
			<td>Included in the Diaplate kit</td>
		</tr>
		<tr>
			<td>Thaumatin from Thaumatococcus daniellii</td>
			<td>Sigma Aldrich</td>
			<td>T7638</td>
			<td>Lyophilized protein</td>
		</tr>
		<tr>
			<td>UV cover film</td>
			<td>SWISSCI</td>
			<td>n/a</td>
			<td>Included in the Diaplate kit</td>
		</tr>
	</tbody>
</table>

high-throughput protein crystallization via microdialysis

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug discovery. To date, X-ray crystallography remains the most successful method for solving three-dimensional protein structures at atomic resolution. With recent advances in serial crystallography, either using X-ray free electron lasers (XFELs) or synchrotron light sources, protein crystallography has progressed to the next frontier, where the ability to acquire time-resolved data provides important mechanistic insights into the behavior of biological molecules at room temperature. This protocol describes a straightforward high-throughput (HTP) workflow for screening crystallization conditions through the use of a 96-well dialysis plate. These plates follow the Society for Biomolecular Screening (SBS) standard and can be easily set up using any standard crystallization laboratory. Once optimal conditions are identified, large quantities of crystals (hundreds of microcrystals) can be produced using the dialyzer. To validate the robustness and versatility of this approach, four different proteins were crystallized, including two membrane proteins.

Four proteins were crystallized using the microdialysis plate (with 10 kDa MWCO membranes), including two membrane proteins. The chicken egg-white lysozyme from the lyophilized powder (see Table of Materials) was prepared at 50 mg.mL-1 in 20 mM NaOAc (pH 4.5), and the lyophilized thaumatin (see Table of Materials) was dissolved in water to a final concentration of 25 mg.mL-1. The two membrane proteins used in this study were the E. coli multidrug efflux pump AcrB and the E. coli lactose transporter&#160;LacY. AcrB was expressed using C43 (DE3) cells and purified by Ni-NTA affinity chromatography and size-exclusion chromatography in 10 mM Tris (pH 7.5), 300 mM NaCl, 0.03% (w/v) n-dodecyl-&#946;-D-maltoside (DDM), and 5% (v/v) glycerol32. Subsequently, the protein was concentrated using a 100 kDa MWCO centrifugal concentrator to a final concentration of 6 mg.mL-1. LacY was also expressed in C43 (DE3) cells, purified by Ni-sepharose affinity chromatography followed by size-exclusion chromatography in 20 mM Tris (pH 7.5), 150 mM NaCl, 0.03% (w/v) DDM, and concentrated using a 100 kDa MWCO concentrator to 10 mg.mL-1 4,33.
For the lysozyme and thaumatin proteins, the 96-well microdialysis plate was filled with a crystallization grid screen prepared in-house as the dialysis solution, while for the membrane protein AcrB, a grid screen was prepared in-house from a previously published crystallization condition34. For LacY, initial hit conditions were found from a commercial screen (see Table of Materials) and further optimized with a grid screen made in-house. The crystallization drop ratio for lysozyme, thaumatin, and AcrB was 1:100, with 2 &#181;L of protein to 200 &#181;L of precipitant (dialysis solution). LacY crystallization drops were also at a 1:100 ratio, with 1 &#181;L of protein to 100 &#181;L of dialysis solution, due to a lower amount of available protein.
Crystals from all four proteins, including the membrane proteins, started to appear between 1-8 h at 20 &#176;C following setup with the microdialysis plate. In this case, it was not necessary to supplement the dialysis solution with detergent for the membrane protein crystallization, typically done during the standard dialysis process to prevent membrane protein aggregation, as the detergent micelles were expected to be larger than the MWCO of the dialysis membranes35.
Following the successful crystallization of the proteins on the microdialysis plate (Figure 4), crystallization conditions were noted, and large-scale crystallization by dialysis was performed using the dialyzer tube with the same 10 kDa MWCO dialysis membranes. Thousands of microcrystals for each of the individual proteins grew inside the dialyzer, as shown in Figure 5. For thaumatin crystallization, 250 &#181;L of protein was loaded onto the dialyzer and dialyzed against 50 mL (1:200 ratio). For AcrB, 250 &#181;L of protein was dialyzed against 25 mL of dialysis solution (1:100). LacY crystallization was also set up at the same ratio with 100 &#181;L of protein to 10 mL of dialysis solution.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64744/64744fig04.jpg" /> 
Figure 4: Crystals are grown by dialysis using the microdialysis plate. The protocol demonstrates the applicability of the setup with soluble proteins: (A) Lysozyme was dialyzed against 0.1 M NaOAc (pH 4.0), 0.5 M NaCl, and 25% (v/v) glycerol. (B) Thaumatin was dialyzed against 0.1 M Bis-tris propane (pH 6.6), 1 M K/Na tartrate, and 18% (v/v) ethylene glycol. Crystals were also obtained for membrane proteins: (C) AcrB was dialysed against 0.1 M MES (pH 5.5), 0.3 M NaCl, and 20% (v/v) PEG-400. (D) LacY was dialysed against 0.1 M MES (pH 6.5), 0.1 M NaCl, and 32% (v/v) PEG-300. Images were captured using a stereo high-magnification microscope with a cross polariser. Scale bars: (A,B,D) = 1 mm; (C) = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64744/64744fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64744/64744fig05.jpg" /> 
Figure 5: Representative images of membrane protein crystals grown by dialysis using the dialyzer tube. (A) Image showing the tube full of microcrystals (indicated by the black arrow). (B) Microscopic image of 2 &#181;L from a slurry of thaumatin crystals grown by dialysis using the 0.5 mL dialyzer tube, dialyzed against 0.1 M Bis-Tris propane (pH 6.6), 1.4 M K/Na tartrate, and 18% (v/v) ethylene glycol. (C) Dark-field (left) and bright-field (right) images of AcrB microcrystals grown by dialysis against 0.1 M MES (pH 5.5), 0.3 M NaCl, and 20% (v/v) PEG-400. (D) Dark-field (left) and bright-field (right) images of LacY microcrystals grown by dialysis against 0.1 M MES (pH 6.5), 0.1 M NaCl, and 32% (v/v) PEG-300. Some precipitates are observed. Scale bars: (B,D) = 200 &#181;m; (C) = 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64744/64744fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High-Throughput Protein Crystallization via Microdialysis at JoVE.com

High-Throughput Protein Crystallization via Microdialysis

The presented protocol describes a straightforward approach for screening protein crystallization conditions and crystal growth using a 96-well high-throughput dialysis plate. The use of dialyzer tubes for the large-scale growth of microcrystals is also demonstrated for serial crystallography and MicroED applications.

High-Throughput Protein Crystallization via Microdialysis

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug ...

high-throughput-protein-crystallization-via-microdialysis

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JoVE Journal

Biochemistry

2.0K Views.  National Physical Laboratory. The presented protocol describes a straightforward approach for screening protein crystallization conditions and crystal growth using a 96-well high-throughput dialysis plate. The use of dialyzer tubes for the large-scale growth of microcrystals is also demonstrated for serial crystallography and MicroED applications.

Video: High-Throughput Protein Crystallization via Microdialysis

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 &#197; resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin&#8211;mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin&#8211;mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin&#8211;mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR&#8211;G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.

This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies.

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Author Spotlight: High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography  

This protocol details high-throughput crystallization screening, ranging from the 1,536 microassay plate preparation to the end of a 6 week experimental time window. Details are included about the sample setup, the imaging obtained, and how users can perform analyses using an artificial intelligence-enabled graphical user interface to quickly and efficiently identify macromolecular crystallization conditions.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

Protein Sample Setup and Crystallization

Crystallization of ABC Transporter Membrane Proteins in Lipidic Bicelles

ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an aqueous environment is typically achieved by employing detergents. These detergents disintegrate the lipid bilayer and solubilize the proteins. The intrinsic habitat of membrane proteins within the lipid bilayer poses a challenge in maintaining their stability and uniformity in solution for structural characterization. Bicelles, which comprise a blend of long and short-chain phospholipids and detergents, replicate the natural lipid structure. The utilization of lipid bicelles and detergents serves as a suitable model system for obtaining high-quality diffraction crystals, specifically to determine the high-resolution structure of membrane proteins. Through these synthetic microenvironments, membrane proteins preserve their native conformation and functionality, facilitating the formation of three-dimensional crystals. In this approach, the detergent-solubilized heterodimeric ABCG5/G8 was reintegrated into DMPC/CHAPSO bicelles, supplemented with cholesterol. This setup was employed in the vapor diffusion experimental procedure for protein crystallization.

Author Spotlight: A Bicelle Crystallization Setup for ABC Transporter Membrane Proteins to Advance Drug Development 

This protocol describes a setup for the crystallization of the sterol transporter ABCG5/G8. ABCG5/G8 is reconstituted into bicelles for hanging-drop crystallization. The protocol does not require specialized materials or substrates, making it accessible and easy to adapt in any laboratory for determining the protein structure through X-ray crystallography.

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an ...