This work was supported by Bio-ID Center and SJTU Cross-Disciplinary Research Fund in Medicine and Engineering (YG2016QN66), National Natural Science Foundation of China (31271217), and National Basic Research Program of China (2014CB910304).

NOTE: The HEK-293T cell line continuously expressing FLAG-tagged hEAG1 channel protein is constructed by transfecting a pCDH lentiviral plasmid containing the DNA sequence of hEAG1 with a FLAG at the distal C-terminus into HEK-293T cells followed by the puromycin-resistant selection as previously described15.
1. Affinity Purification of FLAG-tagged hEAG1 Channel Protein from HEK-293T Cells
<ol>
	<li>Thaw cells stably expressing hEAG1 channels from liquid nitrogen into the warm water (37 &#176;C) quickly. Seed the cells (about 5 x 106 frozen cells) in a 10 cm dish. Grow the cell overnight in Dulbecco&#39;s Modified Eagle&#39;s (DMEM) medium supplemented with 8 mL 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 &#956;g/mL streptomycin, 4 mM L-glutamine and changed the medium the next day.</li>
	<li>Check the GFP fluorescence of these HEK-293T cells by using a fluorescent microscope to make sure the high percentage of cells stably express hEAG1 channels in the culturing system. Exponentially trypsinize the growing cells with 1 mL of 0.25% trypsin for 1 min at room temperature and thenadd 2 mL of serum-containing culture fluid to terminate the digestion. Transfer 400 &#956;L of cell suspension to a 15 cm dish containing 15 mL of complete DMEM medium. Prepare four 15 cm dishes in total.</li>
	<li>Harvest these cells after 2&#8211;3 days culture when the cells reach at about 90% confluency.
	<ol>
		<li>Remove the growth medium from the cells and wash them twice with 4 mL of phosphate buffered saline (1x PBS, pH = 7.4).</li>
		<li>Discard PBS after washing.</li>
		<li>Scrape the cells into 2 mL 1x PBS for each dish by using cell scrape and transfer the scraped cells with 1 mL pipette into a 15 mL tube.</li>
		<li>Centrifuge the cell suspension for 5 min at 420 x g at 4 &#176;C.</li>
		<li>Decant and discard the supernatant.</li>
		<li>Resuspend the cell pellet in total 4 mL of lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM NaCl, 1% NP-40, pH 7.0, contained complete protease inhibitor) for 30 min on ice, and vortex thoroughly the lysate every 10 min.</li>
		<li>Centrifuge the cell lysate for 10 min at 12,000 x g at 4 &#176;C.</li>
		<li>Transfer the supernatant to a 5 mL tube and keep it on ice for immediate use.</li>
	</ol>
	</li>
	<li>Prepare the anti-FLAG M2 affinity resin.
	<ol>
		<li>Thoroughly suspend the resin (supplied as a 50% suspension in store buffer) by gentle inversion to make sure the bottle of anti-FLAG M2 affinity gel is a uniform suspension of gel beads.</li>
		<li>Immediately transfer 400 &#956;L of suspension to a chilled 1.5 mL tube.</li>
		<li>Centrifuge the suspension for 30 s at 8, 000 x g at 4 &#176;C and discard the supernatant carefully to wash out the store buffer.</li>
		<li>Add 500 &#956;L 1x PBS to the resin and suspend the pellet with 1 mL pipette. Then centrifuge the suspension for 30 s at 8, 000 x g at 4 &#176;C and discard the supernatant PBS carefully. Repeat these wash step to clear the stored buffer.</li>
	</ol>
	</li>
	<li>Add 500 &#956;L protein extract supernatant prepared at step 1.3.8 to the resin pellet to suspend the pellet and transfer the suspension to a new chilled 5 mL tube. Repeat this step again to make sure no gel beads left. Add the left protein extract to the mixture.</li>
	<li>Incubate the mixture overnight on a shaker at 8 rpm at 4 &#176;C to capture the FLAG fusion protein.</li>
	<li>Centrifuge the mixture after 12 h incubation for 10 min at 1, 000 x g at 4 &#176;C.</li>
	<li>Discard the supernatant and wash the pellet three times with 500 &#956;L of 1x PBS. Keep it on ice for immediate use.</li>
	<li>Elution the FLAG hEAG1 protein with 3x FLAG peptide.
	<ol>
		<li>Prepare 3X FLAG elution solution. Dissolve 3X FLAG peptide in 500 &#956;L stock solution (0.5 M Tris-HCl, 1 M NaCl, pH = 7.5) at a concentration of 8 &#956;g/&#956;L.</li>
		<li>Add 10 &#956;L 3x FLAG elution solution to 390 &#956;L of PBS to be a 200 ng/&#956;L final concentration solution.</li>
		<li>Add 400 &#956;L of 3x FLAG elution solution to the gel beads prepared at step 1.8.</li>
		<li>Incubate the sample at the shaker at 8 rpm for 2 h at 4 &#176;C.</li>
		<li>Centrifuge the resin for 30 s at 8, 000 x g.</li>
		<li>Transfer the supernatant to a fresh 1.5 mL tube and store it at 4 &#176;C for immediate use.</li>
	</ol>
	</li>
</ol>
2. Concentration Assay and Confirmation of Purified FLAG Fusion hEAG1 by BCA Protein Assay Kit and Western Blotting
<ol>
	<li>Determine the concentration of purified protein using the BCA protein assay kit according the manufacture&#39;s instruction.</li>
	<li>Use 30 &#956;L sample for western analysis to confirm the interest protein had been purified by using an ANTI-FLAG antibody as previously described15.</li>
</ol>
3. Labeling the Purified Channel Protein with Biotin for the BLI Assay
<ol>
	<li>Prepare 5 mg/mL biotin stock solution in PBS. For each test (two biosensors), add a 3-fold molar excess of biotin to 20 &#956;g purified protein to achieve a preferable N-terminal biotinylation of the purified protein in PBS.</li>
	<li>Incubate the sample in the dark on ice for at least 30 min.</li>
	<li>Prepare the sample dilution (SD) buffer: PBS with 0.02% polysorbate 20 and 0.1% bovine serum albumin (BSA, pH 7.4).</li>
	<li>Perform ultrafiltration to change the buffer of the purified channel protein to SD buffer. Remove the unbound biotin by using ultrafiltration device with molecular weight cutoff of 30 kDa, adding the SD buffer and centrifuging the sample at 12,000 x g, for 10 min at 4 &#176;C.</li>
	<li>Remove the ultrafiltrate from the centrifuge tube of ultrafiltration device, add 200 &#956;L SD buffer into the filter device and centrifuging the sample at 12,000 x g, for 10 min at 4&#176;C. Repeat this operation at least three times.</li>
	<li>To collect the buffer exchanged sample, reversed insert the filter device into a 1.5 mL tube and centrifuging them at 2,000 x g, for 5 min at 4 &#176;C. Keep the sample on ice for immediate use.</li>
</ol>
4. Preparation of PIP2 Solution for Assay
<ol>
	<li>Prepare the stock solution of PIP2 (1 mM) in deionized H2O by sonicating for 30 min on ice as previously described17. Store the solution in glass vials at -20 &#176;C and dilute it to the final concentrations immediately before experiments by vigorous vortexing.</li>
</ol>
5. BLI Assay
<ol>
	<li>Turn on the equipment and check the &#34;instrument status&#34; window to confirm the machine is at &#34;ready&#34; state to prewarm the equipment at least for 30 min before the BLI study.</li>
	<li>Make sure the door of the instrument is closed before opening the Data Acquisition software and choose the &#34;New Kinetics Experiment&#34; in the Experiment Wizard.</li>
	<li>Define the wells to be used on the 96-well plate by right click to choose buffer, load, and sample. For sample wells, the unit of concentration of biotinylated FLAG fusion hEAG1 protein should be input as molar (10 &#956;g, 0.09 &#956;M).</li>
	<li>Define the assay steps including baseline, loading, association and dissociation. Choose an assay step and double click on the respective column. A duplicate of assay definition is set for control sensors. Set the rpm as 1,000. Choose 1 min for baseline step and 5&#8211;10 min for loading, association, and dissociation, respectively. Perform the test at room temperature (about 24 &#176;C). 
	NOTE: There are two main procedures: loading the biotinylated channel protein to the sensors and assaying the interaction with small molecule compounds. They can be proceeded continuously (baseline, loading, baseline, association and dissociation). Alternatively, they can be proceeded separately to avoid wasting the test compounds when the first loading part unsuccessful.</li>
	<li>Click the columns which contain the sensors and click the &#34;Fill&#34; to indicate the locations of sensors in the sensor tray.</li>
	<li>Review all planned steps to check for mistakes and go back to correct them.</li>
	<li>Set the location of data files and click &#34;Go&#34; to start the assay. 
	NOTE: The sensors need prewetted for at least 10 min in SD buffer, if this step has been done, then the &#34;Delayed experiment start&#34; setting should be skipped. If not, set a 600 s delay before prewetting the sensors.</li>
	<li>Put a black 96-well plate in the bottom of the tray and insert the A1 corner of the plate into the notch on the tray to seat the plate. For the wells to load the sensors, 200 &#956;L of assay buffer per well add into 2 wells in row A and 2 wells in row B of the 96-well plate.</li>
	<li>Prepare another black 96-well plate as the sample plate and fill the wells with 200 &#956;L SD buffer in row B as control or biotinylated hEAG1 protein solution (10 &#956;g) in row A as assigned during programming in step 5.3. 
	NOTE: Avoid introducing bubbles.</li>
	<li>Open the door of the instrument and insert the sensor tray and sample plate into the left and right plate holder, respectively. Check that the sensor tray and sample plate are positioned correctly based on the shape of left plate holder and the &#34;A1&#34; marker on the top right corner of right plate holder. Close the door and start the assay.</li>
</ol>
6. Data Analysis
<ol>
	<li>Open the Data Analysis software and load the folder containing the assay data. Click &#34;Processing&#34; to get into the processing menu interface and we can see the colorful raw kinetic curves.</li>
	<li>Under Step1: &#34;Data Selection&#34;, click &#34;Sensor Selection&#34;. On the &#34;Sensor Tray #1&#34;, click the sensor wells only wetted with SD buffer and right click to &#34;Change Sensor Type&#34; to &#34;Reference Sensor&#34;. On the &#39;Sample Plate Map&#34;, designate the all the non-specific binding wells and right click to &#34;Change Well Type&#34; to &#34;Reference Well&#34;.</li>
	<li>Tick in the box before &#34;Subtraction&#34; of Step 2 and point &#34;Double Reference&#34;.</li>
	<li>In Step 3: &#34;Align Y Axis&#34;, select &#34;Baseline&#34; as the alignment step. For &#34;Time Range&#34;, enter the last 10 s of that baseline (i.e. From: 0.1 To: 59.8).</li>
	<li>In Step 4: &#34;Inter-step Correction&#34;, select &#34;align to Baseline&#34; to minimize signal shifts between the association and dissociation steps.</li>
	<li>In Step 5: &#34;Process&#34;, select Savitzky-Golay filtering function in most cases and proceed &#34;Process Data&#34;.</li>
	<li>Save Raw Data for further data analysis using other software in Step 7: &#34;Save Results&#34;.</li>
	<li>Click &#34;Analysis | Curve Fitting&#34;.
	<ol>
		<li>For &#34;Step to Analyze&#34;, choose &#34;Association | Dissociation&#34;. For &#34;Model&#34;, select 1:1. 
		NOTE: we choose 1:1 model because it fitted well on our original data and avoided the possibility of over fitting under 1:2 or 2:1 model due to their high freedom. But other options are available here and can be suitably chosen for other fitting analysis for different binding models of analyte.</li>
		<li>For &#34;Fitting&#34;, choose Global (Full). For &#34;Group By&#34;, select &#34;Color&#34;. Select &#34;Rmax Unlinked By Sensor&#34; to allow independent fitting of maximal signal response (Rmax).</li>
		<li>Click &#34;Fit Curves!&#34; to start the nonlinear regression analysis. Hill equation and single exponential function were used in our study.</li>
		<li>Click &#34;Data Export |Save Report&#34; to save fitting results. Or click &#34;Data Export | Export Fitting Results&#34; to save the raw data for further graphing and data analysis with other software.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Membrane ion channels have been verified as the primary therapeutic targets of over 13% of currently known drugs for the treatment of a variety of human diseases, including cardiovascular and neurological disorders18. Patch-clamp recording, the golden standard for measuring the functional of ion channels with small molecules, has been widely used for ion channel ligands screening. However, such electrophysiological approaches cannot demonstrate whether the small molecules binds to the channel dire...

Targeting the cell surface-accessible ion channel proteins with small molecules offers a tremendous potential for the ligand screening and biological drug discovery1,2,3. Thus, an appropriate tool is needed for studying the interaction between ion channel and&#160;small molecules and their corresponding function. The patch-clamp recording has been demonstrated to be a unique and irreplaceable technique in ion channel functional assay. However, determining whether the small molecules directly target ion channels require other technologies. Traditionally, the radioactive ligand binding assay was used to observe the kinetics of binding between small molecule and its target ion channel protein. However, the usage of this technique is limited because of its requirement in radioactive labeling and detection. Moreover, the prerequisite step to label the small ligand in the study prevents its using in many types of ion channels without known specific ligand. Some label-free techniques such as NMR spectroscopy, X-ray diffraction, microscale thermophoresis (MST)4 and surface plasmon resonance (SPR) have been used to measure the protein-small molecule interactions. But these types of assays usually cannot provide sufficient information because of the difficulty to get the full-length protein, low resolution of dynamics, low throughput, and high cost5. In contrast with these techniques, bio-layer Interferometry (BLI) is emerging as a novel label-free methodology to overcome these drawbacks for detecting protein-small molecule interactions by immobilizing a tiny amounts of protein sample on the surfaces of biosensor and measuring the optical changing signals6,7. As a promising biosensor platform, BLI technique is already performed to observe the interaction of small molecules with natural water soluble proteins such as a human monoclonal antibody CR80208 and the detailed assay procedure has been reported in a previous article9. Although the key role of ion channel protein for new therapeutic targets discovery has been recognized, the ion channel protein-small molecule interaction assay based on BLI has not been described.
The human Ether &#224; go-go channels (hEAG1) are expressed in various types of cancer cells and central nervous system which makes the channel a potential therapeutic target of many cancers and neuronal disorders10,11,12,13,14. The electrophysiological study in our lab has confirmed the inhibitory effect of phosphatidylinositol 4, 5-bisphosphate (PIP2) on hEAG1 channel15. Based on our results, testing PIP2 directly interaction with the hEAG1 by using BLI technique can be as a model for other types of ion channel protein-small molecule compound interaction especially for those channels lacking specific ligands. According to the instructions of BLI assay, we prepared biotinylated hEAG1 proteins and immobilized them on the surface of streptavidin (SA) biosensor tips followed by interaction them to PIP2 solutions to observe their direct binding between the protein and the lipid. After the attachment of PIP2 to the hEAG1 protein coated surface, the thickness of the layer on the surface increases, which directly correlates the spectral shift and can be measured in real-time16. The binding kinetics can be determined due to a positive shift in association step and a negative shift in dissociation step. According to this principle, we purified the functional hEAG1 ion channel protein from HEK-239T stable expression system by using affinity purification method to maintain the in vitro functional state, then measured the kinetics of binding of different concentration PIP2, and yielded a semblable kinetic data as observed in electrophysiological measurements15. The close correspondence between the results from the BLI and electrophysiological measurements demonstrate for the first time the suitability of BLI as an appropriate analytical tool for ion channel membrane protein-small molecule interaction.

<table>
	<tbody>
		<tr>
			<td>DMEM/High Glucose Medium</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1x)</td>
			<td>HyClone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>1697550</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Corning</td>
			<td>430599</td>
			<td>150 mm X 25 mm</td>
		</tr>
		<tr>
			<td>Nonidet P-40 Substitute</td>
			<td>Amresco</td>
			<td>E109</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>BBI Life Sciences</td>
			<td>A610476</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>BBI Life Sciences</td>
			<td>A610440</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>BBI Life Sciences</td>
			<td>A600332</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene-20-Sorbitan Monolaurate</td>
			<td>BBI Life Sciences</td>
			<td>A600560</td>
			<td></td>
		</tr>
		<tr>
			<td>ANTI-FLAG M2 Affinity Gel</td>
			<td>Sigma</td>
			<td>A2220</td>
			<td></td>
		</tr>
		<tr>
			<td>3x FLAG peptide</td>
			<td>Sigma</td>
			<td>F4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Octet-RED96</td>
			<td>Pall/Fort&#233;Bio</td>
			<td>30-5048</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition software</td>
			<td>Pall/Fort&#233;Bio</td>
			<td>Version 7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Analysis software</td>
			<td>Pall/Fort&#233;Bio</td>
			<td>Version 7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosensor/Streptavidin</td>
			<td>Pall/Fort&#233;Bio</td>
			<td>18-5019</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtiter plate</td>
			<td>Greiner Bio-one</td>
			<td>655209</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-NHS-LC-LC-Biotin</td>
			<td>ThermoFisher</td>
			<td>21338</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal Machine</td>
			<td>ThermoFisher</td>
			<td>75004250</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder</td>
			<td>ThermoScientific</td>
			<td>318120</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrafiltration device</td>
			<td>MILLIPORE</td>
			<td>UFC503008</td>
			<td>NMWL of 30 kDa</td>
		</tr>
		<tr>
			<td>phosphatidylinositol 4, 5-bisphosphate (PIP2)</td>
			<td>Sigma</td>
			<td>P9763</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal ANTI-FLAG M2 antibody</td>
			<td>Sigma</td>
			<td>F1804</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>goat anti-mouse HRP-conjugated secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
			<td>1:5000 dilution</td>
		</tr>
		<tr>
			<td>Enhanced BCA Protein Assay Kit</td>
			<td>Beyotime</td>
			<td>P0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>04693159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Imager 600 Imaging System</td>
			<td>GE Healthcare Bio-Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Western blot system</td>
			<td>BIO-RAD</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

capturing the interaction kinetics of an ion channel protein with small molecules by the bio-layer interferometry assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

We purified&#160;the FLAG fusion hEAG1 channel protein from HEK-293T cells stably overexpressed hEAG1. The function of this fusion protein has been demonstrated by using the patch-clamp method and the quality and specificity of purified protein are confirmed by Western blot (Figure 1). The purified channel protein is biotinylated to perform an interaction assay with the lipids (PIP2) by using the real-time BLI assay. The BLI binding assay configura...

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Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

capturing-interaction-kinetics-an-ion-channel-protein-with-small

Research

JoVE Journal

Biochemistry

8.2K Views.  Shanghai Jiao Tong University School of Medicine. Het protocol hier beschrijft de interacties van gezuiverde hEAG1 ion kanaal eiwit met de kleine molecuul lipide ligand phosphatidylinositol 4, 5-difosfaat (PIP2). De meting toont aan dat BLI zou een mogelijke methode voor nieuwe kleine-molecuul ion kanaal ligand screening.

Video: Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

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 ...

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. Although the molecular mechanisms for the biological responses to fluid flow/shear force are not fully understood, fluid flow-mediated regulation of ion channel gating may contribute critically. Therefore, fluid flow/shear force sensitivity of ion channels has been studied using the patch-clamp technique. However, depending on the experimental protocol, the outcomes and interpretation of data can be erroneous. Here, we present experimental and theoretical evidence for fluid flow-related errors and provide methods for estimating, preventing, and correcting these errors. Changes in junction potential between the Ag/AgCl reference electrode and bathing fluid were measured with an open pipette filled with 3 M KCl. Fluid flow could then shift the liquid/metal junction potential to approximately 7 mV. Conversely, by measuring the voltage shift induced by fluid flow, we estimated the ion concentration in the unstirred boundary layer. In the static condition, the real ion concentrations adjacent to the Ag/AgCl reference electrode or ion channel inlet at the cell-membrane surface can reach as low as approximately 30% of that in the flow condition. Placing an agarose 3 M KCl bridge between the bathing fluid and reference electrode may have prevented this problem of junction potential shifting. However, the unstirred layer effect adjacent to the cell membrane surface could not be fixed in this way. Here, we provide a method for measuring real ion concentrations in the unstirred boundary layer with an open patch-clamp pipette, emphasizing the importance of using an agarose salt-bridge while studying fluid flow-induced regulation of ion currents. Therefore, this novel approach, which takes into consideration the real concentrations of ions in the unstirred boundary layer, may provide useful insight on the experimental design and data interpretation related to fluid shear stress regulation of ion channels.

Mechanosensitive ion channels are often studied in terms of fluid flow/shear force sensitivity with patch-clamp recording. However, depending on the experimental protocol, the outcome on fluid flow-regulations of ion channels can be erroneous. Here, we provide methods for preventing and correcting such errors with a theoretical basis.

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

This article presents a method to generate protein crystals derivatized with I3C (5-amino-2,4,6-triiodoisophthalic acid) using microseeding to generate new crystallization conditions in sparse matrix screens. The trays can be set up using liquid dispensing robots or by hand.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

Microcrystal Electron Diffraction of Small Molecules

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.

Here, we describe the procedures developed in our laboratory for preparing powders of small molecule crystals for microcrystal electron diffraction (MicroED) experiments.

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

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