Name: extracellular protein microarray technology for high throughput detection of low affinity receptor-ligand interactions
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions at JoVE.com

We thank Philamer Calses and Kobe Yuen for critically reading the manuscript. We are thankful to Randy Yen for excellent technical advice.

1. Generation of a Library of Extracellular Human Proteins
<ol>
	<li>Compile a list of cell surface receptors or secreted proteins of interest to build the protein microarray library. Specific protein families (for example, the immunoglobulin superfamily) or proteins selectively expressed in particular cell types can be selected for the study.</li>
	<li>For cell surface receptors, determine the extracellular domain (ECD) boundaries by identifying the signal peptide and transmembrane regions using software tools. Some o...

<ol>
	<li>Overington, J. P., Al-Lazikani, B., Hopkins, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+many+drug+targets+are+there?.">How many drug targets are there?.</a> Nature Reviews Drug Discovery. 5 (12), 993-996 (2006).</li><li>Wright, G. J., Martin, S., Bushell, K. M., Sollner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+identification+of+transient+extracellular+protein+interactions.">High-throughput identification of transient extracellular protein interactions.</a> Biochemical Society Transactions. 38 (4), 919-922 (2010).</li><li>Wright, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+initiation+in+biological+systems:+the+properties+and+detection+of+transient+extracellular+protein+interactions.">Signal initiation in biological systems: the properties and detection of transient extracellular protein interactions.</a> Molecular BioSystems. 5 (12), 1405-1412 (2009).</li><li>Ramani, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+secreted+protein+microarray+platform+for+extracellular+protein+interaction+discovery.">A secreted protein microarray platform for extracellular protein interaction discovery.</a> Analytical Biochemistry. 420 (2), 127-138 (2012).</li><li>Tom, I., Lewin-Koh, N., Ramani, S. R., Gonzalez, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+microarrays+for+identification+of+novel+extracellular+protein-protein+interactions.">Protein microarrays for identification of novel extracellular protein-protein interactions.</a> Current Protocols in Protein Science. 27, 1-27 (2013).</li><li>Kaushansky, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+protein-protein+interactions+in+high+throughput+using+protein+domain+microarrays.">Quantifying protein-protein interactions in high throughput using protein domain microarrays.</a> Nature Protocols. 5 (4), 773-790 (2010).</li><li>Popescu, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+binding+of+calmodulin-related+proteins+to+their+targets+revealed+through+high-density+Arabidopsis+protein+microarrays.">Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (11), 4730-4735 (2007).</li><li>Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+protein+activities+using+proteome+chips.">Global analysis of protein activities using proteome chips.</a> Science. 293 (5537), 2101-2105 (2001).</li><li>Bushell, K. M., Sollner, C., Schuster-Boeckler, B., Bateman, A., Wright, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+screening+for+novel+low-affinity+extracellular+protein+interactions.">Large-scale screening for novel low-affinity extracellular protein interactions.</a> Genome Research. 18 (4), 622-630 (2008).</li><li>Voulgaraki, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivalent+recombinant+proteins+for+probing+functions+of+leucocyte+surface+proteins+such+as+the+CD200+receptor.">Multivalent recombinant proteins for probing functions of leucocyte surface proteins such as the CD200 receptor.</a> Immunology. 115 (3), 337-346 (2005).</li><li>Jiang, L., Barclay, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+leucocyte+surface+protein+interactions+by+high-throughput+screening+with+multivalent+reagents.">Identification of leucocyte surface protein interactions by high-throughput screening with multivalent reagents.</a> Immunology. 129 (1), 55-61 (2010).</li><li>Yeh, F. L., Wang, Y., Tom, I., Gonzalez, L. C., Sheng, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM2+Binds+to+Apolipoproteins,+Including+APOE+and+CLU/APOJ,+and+Thereby+Facilitates+Uptake+of+Amyloid-Beta+by+Microglia.">TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia.</a> Neuron. 91 (2), 328-340 (2016).</li><li>Jaworski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operational+redundancy+in+axon+guidance+through+the+multifunctional+receptor+Robo3+and+its+ligand+NELL2.">Operational redundancy in axon guidance through the multifunctional receptor Robo3 and its ligand NELL2.</a> Science. 350 (6263), 961-965 (2015).</li><li>Martinez-Martin, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+interactome+of+the+human+adenovirus+family+reveals+diverse+strategies+for+immunomodulation.">The extracellular interactome of the human adenovirus family reveals diverse strategies for immunomodulation.</a> Nature Communications. 7, 11473 (2016).</li><li>Nielsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+Secretory+Proteins+with+SignalP.">Predicting Secretory Proteins with SignalP.</a> Methods in Molecular Biology. 1611, 59-73 (2017).</li><li>Krogh, A., Larsson, B., von Heijne, G., Sonnhammer, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+transmembrane+protein+topology+with+a+hidden+Markov+model:+application+to+complete+genomes.">Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.</a> Journal of Molecular Biology. 305 (3), 567-580 (2001).</li><li>Kall, L., Krogh, A., Sonnhammer, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+combined+transmembrane+topology+and+signal+peptide+prediction--the+Phobius+web+server.">Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server.</a> Nucleic Acids Research. 35 (Web Server issue), W429-W432 (2007).</li><li>Bernsel, A., Viklund, H., Hennerdal, A., Elofsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TOPCONS:+consensus+prediction+of+membrane+protein+topology.">TOPCONS: consensus prediction of membrane protein topology.</a> Nucleic Acids Research. 27 (Web Server issue), W465-W468 (2009).</li><li>Gonzalez, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+the+mammalian+extracellular+proteome+for+regulators+of+embryonic+human+stem+cell+pluripotency.">Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (8), 3552-3557 (2010).</li><li>Durocher, Y., Perret, S., Kamen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+and+high-throughput+recombinant+protein+production+by+transient+transfection+of+suspension-growing+human+293-EBNA1+cells.">High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells.</a> Nucleic Acids Research. 30 (2), E9 (2002).</li><li>Battle, T., Antonsson, B., Feger, G., Besson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+mammalian+protein+expression,+purification,+aliquoting+and+storage+pipeline+to+assemble+a+library+of+the+human+secretome.">A high-throughput mammalian protein expression, purification, aliquoting and storage pipeline to assemble a library of the human secretome.</a> Combinatorial Chemistry &amp; High Throughput Screening. 9 (9), 639-649 (2006).</li><li>Clark, H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secreted+protein+discovery+initiative+(SPDI),+a+large-scale+effort+to+identify+novel+human+secreted+and+transmembrane+proteins:+a+bioinformatics+assessment.">The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment.</a> Genome Research. 13 (10), 2265-2270 (2003).</li></ol>

A significant number of orphan receptors remain in the human genome, and novel interacting partners continue to emerge for extracellular proteins with previously characterized ligands. Defining the receptor-ligand interactions in human and model organisms is essential to understand the mechanisms that dictate cellular communication during homeostasis, as well as dysregulation leading to disease, and therefore inform new or improved therapeutic options. Nevertheless, detection of extracellular protein interactions by wide...

The method reviewed here describes the printing of a collection of extracellular proteins in a microarray format, followed by a method for screening of a target of interest against this library. We have identified protein multimerization as a crucial step for detection of interactions characterized by low binding affinities. To enhance detection of these interactions, we describe a protocol based on multimerization of the query protein of interest using microbeads.
Secreted and cell surface-expressed proteins (collectively termed extracellular proteins) along with their interacting partners are key regulators of cellular communication, signaling and interaction with the microenvironment. They are, therefore, essential in regulating many physiological and pathological processes. Approximately a quarter of the human genome (&#8776;5,000 proteins) encodes for extracellular proteins, which, given their significance and accessibility to systematically delivered drugs, represent key targets for drug development1. Consequently, extracellular proteins represent more than 70% of the protein targets with known pharmacological action for approved drugs on the market, known as the &#34;druggable proteome&#34;. Despite their importance and abundance, the extracellular protein-protein interaction (ePPI) networks remain remarkably underrepresented in the available databases. This is fundamentally due to the complex biochemical nature of the extracellular proteins, which precludes their characterization using most available technologies2. Firstly, membrane proteins are difficult to solubilize, a process that often involves harsh washing conditions and detergents; secondly, extracellular proteins often lack relevant post-translational modifications such as glycosylation that are absent when these proteins are expressed in commonly used heterologous systems. Finally, interactions between receptors, such as co-receptors expressed on immune cells, are often transient and characterized by very low affinities (KD in the ~1 &#956;M to &#62;100 &#956;M range). Altogether, the nature of these proteins and their binding partners render most widely utilized technologies, such as affinity purification/mass spectrometry (AP/MS) or yeast-two-hybrid, unsuitable for detection of interactions in the extracellular space2,3.
In an effort to overcome these technical challenges and accelerate the discovery of novel interactions for extracellular proteins, we have developed a high coverage extracellular protein microarray4,5. Microarrays offer the advantage of generating high-density surfaces with small amounts of sample, and are generally amenable to high throughput studies. Protein microarray-based studies have previously provided relevant insights into protein interactions for several model organisms, albeit mainly focusing on cytosolic interactions or on specific protein families6,7,8. In contrast, limited work has been done to investigate extracellular protein interactions using this technology. We have developed a protein microarray method to enable studies of ePPIs by building a comprehensive and highly diverse library of purified secreted proteins and single transmembrane (STM) receptors expressed as recombinant extracellular domains (ECD) fused to common tags for affinity purification4. The success of the protein microarray screens relies heavily on the establishment of a high quality protein library. For expression of both the library and query protein, mammalian cells or insect cells were preferentially chosen as heterologous expression systems, to ensure proper addition of post-translational modifications such as glycosylation or disulphide bonds. SDS-PAGE, size exclusion chromatography and multi-angle laser light scattering are techniques commonly utilized to assess recombinant protein quality. The protein library is then spotted onto epoxysilane slides and stored at -20 &#176;C for long-term use. Protein concentrations above of 0.4 mg/mL are recommended for the protocol described below. Therefore, low-expressing proteins may require a concentration step prior to sample printing and storage. Nevertheless, a main advantage of this technique is the small volume of protein required (50 &#956;g of protein is sufficient to perform &#62;2,000 screens), alongside minimal query protein consumption (20-25 &#956;g per duplicates screen). Using the protocol and equipment described here, and provided libraries are available, results for individual query proteins can be generated within one working day.
A major challenge in detecting protein interactions in the extracellular environment arises from their characteristically weak or transient nature, which precludes identification by most commonly used methodologies. Increasing the binding avidity greatly improves sensitivity for detection of weak protein interactions9,10,11. Based on this principle we developed a method to multimerize the query proteins (expressed as Fc fusion) using protein A-coated beads4,5. To avoid any potential inactivation of the query protein by random labeling, we instead label an irrelevant human immunoglobulin G with Cy5 and add it along with the query protein to the protein A beads, thus eliminating any artifacts due to the direct conjugation of a dye to the protein of interest. Given the micromolar affinities of several co-receptor pairs, the multivalent complexes greatly enhance signal to noise ratio, compared to Fc-fusion query proteins screened as soluble proteins4.
In summary, the goal of this protocol is to describe the preparation of microarray slides containing a pre-existing extracellular protein library for identification of receptor-ligand interactions. We review the steps for slide printing, followed by a protocol for screening of a protein of interest against the extracellular protein library. Moreover, we describe a method for enhanced detection of ePPIs based on microbeads to achieve increased avidity of the protein under study. The extracellular protein microarray technology described here represents a fast, robust and effective approach for screening and detecting novel ePPI with low false-positive ratios, and by utilizing only microgram quantities of the query protein under investigation. This technology has fueled multiple studies that have provided relevant insights into previously unknown cellular functions and signaling pathways for a variety of receptors12,13, including viral immunoregulators14, and can be utilized to de-orphanize any extracellular protein of interest.

<table>
	<tbody>
		<tr>
			<td>Ultra Pure MB Grade glycerol</td>
			<td>USB Corporation</td>
			<td>56-81-5</td>
			<td>Protein storage</td>
		</tr>
		<tr>
			<td>SeptoMark blocking buffer&#160;</td>
			<td>Zeptosens</td>
			<td>BB1, 90-40</td>
			<td>Blocking buffer microarray slides</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Roche</td>
			<td>03-117-957-001</td>
			<td>Slide control for mask fitting (optional)</td>
		</tr>
		<tr>
			<td>Polypropylene multiwell plates</td>
			<td>Greiner Bio One</td>
			<td>82050-678</td>
			<td>Protein storage</td>
		</tr>
		<tr>
			<td>Polypropylene multiwell plates</td>
			<td>Arrayit</td>
			<td>MMP384</td>
			<td>Slide printing</td>
		</tr>
		<tr>
			<td>NanoPrint LM60, or similar contact microarrayer</td>
			<td>Arrayit</td>
			<td>NanoPrint LM60, or similar contact microarrayer</td>
			<td>Slide printing</td>
		</tr>
		<tr>
			<td>Micro spotting pins</td>
			<td>Arrayit</td>
			<td>Micro spotting pins</td>
			<td>Slide printing</td>
		</tr>
		<tr>
			<td>ZeptoFOG blocking station</td>
			<td>Zeptosens</td>
			<td>ZeptoFOG blocking station, 1210</td>
			<td>Block slides after printing</td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Thermo Fisher</td>
			<td>LP0031</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>Epoxysilane-coated glass slide</td>
			<td>Nextrion Slide E</td>
			<td>1064016</td>
			<td>Microarray slides</td>
		</tr>
		<tr>
			<td>Glass holder and slide rack set</td>
			<td>Wheaton</td>
			<td>900303</td>
			<td>Slide storage</td>
		</tr>
		<tr>
			<td>Cy5 monoreactive dye</td>
			<td>GE Healthcare</td>
			<td>PA23031</td>
			<td>Albumin labeling</td>
		</tr>
		<tr>
			<td>Cy5 monoreactive dye</td>
			<td>GE Healthcare</td>
			<td>PA25001</td>
			<td>Human IgG labeling</td>
		</tr>
		<tr>
			<td>Pro-spin desalting column</td>
			<td>Princeton Separations</td>
			<td>CS-800</td>
			<td>Remove free dye</td>
		</tr>
		<tr>
			<td>Adhesive aluminum foil seal</td>
			<td>AlumaSeal</td>
			<td>F-384-100</td>
			<td>Seal stock plates</td>
		</tr>
		<tr>
			<td>Polypropylene cryogenic vials</td>
			<td>Corning</td>
			<td>430658</td>
			<td>Master vials for protein library storage</td>
		</tr>
		<tr>
			<td>Protein A microbeads</td>
			<td>Miltenyi</td>
			<td>120-000-396</td>
			<td>Query protein multimerization</td>
		</tr>
		<tr>
			<td>Human IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>&#160;009-000-003</td>
			<td>Irrelevant IgG for labeling</td>
		</tr>
		<tr>
			<td>Protein A</td>
			<td>Sigma</td>
			<td>&#160;P7837</td>
			<td>Microarray slide blocking</td>
		</tr>
		<tr>
			<td>Hybridization station, a-Hyb or similar</td>
			<td>Miltenyi</td>
			<td>Hybridization station, a-Hyb or similar</td>
			<td>Automated microarray processing (optional)</td>
		</tr>
		<tr>
			<td>GenePix 4000B scanner or similar</td>
			<td>Molecular Devices</td>
			<td>GenePix 4000B scanner or similar</td>
			<td>Slide scanning</td>
		</tr>
		<tr>
			<td>GenePix Pro or equivalent data extraction software</td>
			<td>Molecular Devices</td>
			<td>GenePix Pro or equivalent data extraction software</td>
			<td>Data processing</td>
		</tr>
		<tr>
			<td>Signal P4.1</td>
			<td>DTU Bioinformatics, Technical University of Denmark</td>
			<td>online software</td>
			<td>Prediction tool to determine presence and location of signal peptide cleavage sites</td>
		</tr>
		<tr>
			<td>TMHMM 2.0 server</td>
			<td>DTU Bioinformatics, Technical University of Denmark</td>
			<td>online software</td>
			<td>Prediction of transmembrane helices in proteins</td>
		</tr>
		<tr>
			<td>Phobius</td>
			<td>Stockholm Bioinformatics Center</td>
			<td>online software</td>
			<td>A combined transmembrane topology and signal peptide predictor</td>
		</tr>
		<tr>
			<td>TOPCONS</td>
			<td>Stockholm University</td>
			<td>online software</td>
			<td>Prediction of membrane topology and signal peptides</td>
		</tr>
	</tbody>
</table>

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.

A schematic of the workflow for the extracellular protein microarray technology is shown in Figure 1. Once the microarray slides containing the extracellular protein library are available, the screening of the protein of interest and data analysis can be completed within one day. Many physiologically relevant interactions between membrane-embedded receptors are characterized by very weak binding strengths (KD in the micromolar range). To improve de...

Watch this Scientific Journal Video about Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions at JoVE.com

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

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 ...

Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

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 ...

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

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 ...

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible ...

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

High-Throughput Protein Crystallization via Microdialysis

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 Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography (Video) | JoVE 

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