We thank Fort&#233;Bio for providing graphics used in Figure 1. This work was supported by NIH grant GM083088 to T.M.D.

1. Programming the Instrument for BLI Assay
Turn on the instrument at least one hour in advance to allow the lamp to warm up; this is necessary to minimize noise and drift in optical signal during the experiment. Set the desired temperature via the instrument tab to prewarm the sample plate holder. Then set up the experimental design in the Data Acquisition software. Select "New Kinetics Experiment" in the Experiment Wizard tab. This presents a tabbed menu with all steps that must be defined.
1.1. Plate Definition
Define the columns to be used on the 96-well sample plate. Assign columns to contain buffer, immobilized protein or binding partner. For each association well, enter the concentration of binding partner to be used. Note: the plate definition shown in Figure 2 has options for distinct assays.
1.2. Assay Definition
Define all steps needed for the assay. These include (i) Baseline (several), (ii) Loading, (iii) Association and (iv) Dissociation of binding partner. Then, as described below, link individual assay steps with columns of wells on the sample plate by selecting the assay step and then double clicking on the respective column. This programs the sensors to be moved from one column of wells to the next during the assay.
<ol>
	<li>Baseline 
		Begin with a brief baseline step (&#8805;60 sec), with sensors in assay buffer (Figure 2, column 1), to establish initial BLI signals in the 96-well plate.</li>
	<li>Loading (immobilizing the biotinylated protein on the sensors) 
		Assign sensors to wells that will contain the biotinylated protein (Figure 2, column 2). Use the threshold function to achieve a predetermined level of binding (see Introduction). Set the threshold option so that all sensors will be moved to the next column of wells when any one of the sensors reaches the threshold.</li>
	<li>Baseline 
		Include another baseline step in buffer (Figure 2, column 3; typically &#62;5 min) to wash away nonimmobilized biotinylated proteins from the sensors and establish new, stable baseline signals. For basic binding/dissociation assays only (as in Figure 3), include an extra baseline step (60 sec) with sensors in new wells of buffer (Figure 2, column 4) before the Association step.</li>
	<li>Association (of the binding partner to the immobilized protein) 
		For a basic binding/dissociation assay (as in Figure 3), select a range of concentrations of binding partner to be used for different sensors/wells (Figure 2, column 5). Adjust the step time so that at least a saturating concentration of the binding partner should approach equilibrium binding signal. For an assay to test allosteric effects of small ligands on dissociation of the protein-protein complex (as in Figure 5), select a single high concentration of binding partner (~10-fold above the estimated KD) for use in all association wells.</li>
	<li>Dissociation (of the binding partner) 
		For a basic binding/dissociation assay only (as in Figure 3), designate the sensors to return to column 4 (Figure 2), the same buffer wells as used for the extra baseline before Association. For an assay to test allosteric effects of small ligands, instead designate sensors to move to column 6 (Figure 2), where each well can contain buffer plus different allosteric ligands.</li>
</ol>
1.3. Sensor Assignment
Indicate the locations in the sensor tray that will contain prewetted sensors for the assay. Identify any empty positions since the experiment will fail if no sensors are picked up.
1.4. Review Experiment
Visualize all planned steps to check for mistakes and go back to correct them.
1.5. Run Experiment
Enter necessary details, including location of data files. Enter the desired temperature for the experiment. If sensors still require prewetting, select the option to delay starting the experiment. Finally, once all sample preparation is complete (step 2) and both the sample plate and sensor tray are loaded in the instrument , click the Go button to run the assay.
2. Sample Preparation
<ol>
	<li>Prepare an appropriate assay buffer. Buffer used for experiments shown in Figures 3 and 5: 20 mM MOPS (3-(N-Morpholino)propanesulfonic acid), Tris (Tris(hydroxymethyl)amino-methane) (added to adjust pH to 8.0), 50 mM KCl. Include BSA (bovine serum albumin, fatty-acid free, 0.5 mg/ml final) in the buffer for all assay steps and for all dilutions of the biotinylated protein or binding partner to minimize nonspecific binding of proteins to the sensors.</li>
	<li>Dilute the biotinylated protein (&#949;) in assay buffer to an appropriate concentration (see Introduction).</li>
	<li>Prepare dilutions of the binding partner (F1(-&#949;)) in assay buffer (see Discussion for range of concentrations to use).</li>
	<li>Prewet the streptavidin-coated sensors for at least 10 min to remove their protective sucrose coating. Remove the sensor-containing rack from the sensor tray and insert a 96-well plate in the bottom of the tray, sliding one corner of the plate into the orienting notch on the tray. For the column of sensors to be used, add 200 &#956;l of assay buffer per well in that column of the 96-well plate. Return the rack of sensors to the tray in the correct orientation (with tabs in slots).</li>
	<li>As assigned during programming in Step 1, fill wells of the sample plate with assay buffer or the appropriate protein dilutions, as in Figure 2. Avoid introducing bubbles, as they can cause noise in the optical signal. Include one or more reference wells that omit either (i) biotinylated protein or (ii) binding partner.</li>
	<li>Open the door of the instrument and insert the sensor tray onto the stage (left), with the tray&#8217;s tabs inserted into the slots of the stage. Insert the sample plate into the plate holder (right); make sure that the plate is seated flat and in the correct orientation, as indicated on the plate holder. Close the door and start the assay from the Data Acquisition program (step 1.5).</li>
</ol>
3. Data Processing
<ol>
	<li>After the assay has run, open the Data Analysis software and load the folder containing the data. Click the "Processing" tab to see a step-wise Processing menu (at left) and the raw kinetic data, with each sensor (A-H) assigned a different color (see Figure 3).</li>
	<li>Under "Data Selection", click the "Sensor Selection" button. On the "Sample Plate Map", designate the wells for 1 or 2 control sensors (G, H in assay of Figure 3) as reference wells. On the Processing menu, check the "Subtraction" box and select "Reference Wells" to subtract reference signal (single or averaged) from every other sensor&#8217;s signal.</li>
	<li>Align all traces to Y=0 by using the "Align Y Axis" step. Select "Baseline" as the alignment step (this being the last baseline before the Association step). For "Time Range", enter the last 10 sec of that baseline (i.e. 50-60 sec for a 60-sec baseline, as in Figure 3).</li>
	<li>Check the "Inter-step Correction" box to minimize signal shifts between the Association and Dissociation steps. Select align to Baseline or Dissociation.</li>
	<li>Select Savitzky-Golay filtering function in most cases and click "Process Data!" to proceed. Visually inspect the final processed data (lower right panel). With assays intended for global data analysis (as in Figure 4), if signal traces show a significant shift between Association and Dissociation steps, change the selection of Dissociation or Baseline for the "Inter-step Correction" and reprocess the data before proceeding to Data Analysis.</li>
</ol>
4. Data Analysis
Click the "Analysis" tab in Data Analysis to begin. Note: the example steps below are for global analysis of multiple binding/dissociation curves (as in Figure 3).
<ol>
	<li>For "Step to Analyze", choose Association and Dissociation. For "Model", select 1:1. Note: other limited options are available.</li>
	<li>For "Fitting", choose Global (Full). For "Group By" select Color (as on the graph). Select "Rmax Unlinked By Sensor" to allow independent fitting of Rmax (maximal signal response upon saturating binding of the partner to the immobilized protein). Note: we do this for most assays, as sensors vary slightly in the amount of protein immobilized (see Figure 3, Loading).</li>
	<li>On the table shown, make sure all sensor traces to be analyzed have the same color, so they will be analyzed as a global set. If needed, select one or more sensor traces to omit from global fitting: under the "Include" column, right-click on the desired sensor position and select "Exclude Wells".</li>
	<li>Click "Fit Curves!" to start the nonlinear regression analysis. Examine the fitting results, which include (i) overlay of regression curves with sensor data traces (as in Figure 4), (ii) plots of fitting residuals, and (iii) a table with determined parameter values (rate constants, Rmax, KD) and statistics (standard errors for parameters, Chi-squared, R2).</li>
	<li>Under "Data Export", save fitting results by clicking "Export Table to .csv File". For graphing or further analysis of data/fitted curves with other software, click "Export Fitting Results" to save each sensor&#8217;s data and fitted curve in a text file.</li>
</ol>

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	<li>Nirschl, M., Reuter, F., Voros, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+Transducer+Principles+for+Label-Free+Biomolecular+Interaction+Analysis.">Review of Transducer Principles for Label-Free Biomolecular Interaction Analysis.</a> Biosensors. 1, 70-92 (2011).</li><li>Homola, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+and+future+of+surface+plasmon+resonance+biosensors.">Present and future of surface plasmon resonance biosensors.</a> Anal. Bioanal. Chem. 377, 528-539 (2003).</li><li>Piehler, J., Brecht, A., Gauglitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+Detection+of+Low+Molecular+Weight+Analytes.">Affinity Detection of Low Molecular Weight Analytes.</a> Anal. Chem. 68, 139-143 (1996).</li><li>Duncan, T. M., Hackney, D. D., Tamanoi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Enzymes.">The Enzymes.</a> Energy Coupling and Molecular Motors Vol. XXIII, 203-275 (2004).</li><li>Feniouk, B. A., Suzuki, T., Yoshida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+subunit+&#949;+in+the+catalysis+and+regulation+of+FOF1-ATP+synthase.">The role of subunit &#949; in the catalysis and regulation of FOF1-ATP synthase.</a> Biochim. Biophys. Acta. 1757, 326-338 (2006).</li><li>Cingolani, G., Duncan, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+ATP+synthase+catalytic+complex+(F1)+from+Escherichia+coli+in+an+autoinhibited+conformation.">Structure of the ATP synthase catalytic complex (F1) from Escherichia coli in an autoinhibited conformation.</a> Nat. Struc. Mol. Biol. 18, 701-707 (2011).</li><li>Mirsaeidi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=After+40+years,+new+medicine+for+combating+TB.">After 40 years, new medicine for combating TB.</a> Int. J. Mycobacteriol. 2, 1-2 (2013).</li><li>Shah, N. B., Hutcheon, M. L., Haarer, B. K., Duncan, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F1-ATPase+of+Escherichia+coli:+The+&#1013;-inhibited+state+forms+after+ATP+hydrolysis,+is+distinct+from+the+ADP-inhibited+state,+and+responds+dynamically+to+catalytic+site+ligands.">F1-ATPase of Escherichia coli: The &#1013;-inhibited state forms after ATP hydrolysis, is distinct from the ADP-inhibited state, and responds dynamically to catalytic site ligands.</a> J. Biol. Chem. 288, 9383-9395 (2013).</li><li>Burgess, T. L., Ross, S. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+plasmid+expression+vector+for+the+production+of+biotinylated+proteins+by+site-specific,+enzymatic+modification+in+Escherichia+coli.">A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli.</a> Gene. 169, 59-64 (1996).</li><li>Dayne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BioLayer+Interferometry+(BLI)+-+How+Does+it+Work?.">BioLayer Interferometry (BLI) - How Does it Work?.</a> Fort&#233;Bio newsletter In Interactions. 5, 8-9 (2012).</li><li>Wartchow, 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=Biosensor-based+small+molecule+fragment+screening+with+biolayer+interferometry.">Biosensor-based small molecule fragment screening with biolayer interferometry.</a> J. 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The authors declare that they have no conflicts of interest.

Currently available instruments for BLI allow significant throughput and flexibility in assays for biomolecular interactions. Various solution samples are dispensed in wells of a black microtiter plate, and a set of parallel BLI sensors are programmed to move back and forth between columns of wells on the plate. The samples are stirred by orbital shaking throughout the assay. The system used here has 8 sensors and uses a 96-well sample plate, but another system uses 16 sensors and a 384-well sample plate. Thus, interacti...

Protein-protein interactions are important for many biological processes, and label-free optical methods like Surface Plasmon Resonance (SPR) have been used in vitro to study kinetics of binding and dissociation1. Most label-free methods immobilize one biomolecule on a sensor surface and use an optical signal to detect a binding partner from solution as it associates with the immobilized biomolecule1. While SPR is a highly sensitive method, it is prone to interference due to changes in the refractive index of the solution flowing over the sensor2. Although not as sensitive as SPR, Bio-layer Interferometry (BLI) is less affected by changes in sample composition1,3. BLI uses fiber optic biosensors that have a proprietary biocompatible coating at the tip. The system used here (Octet-RED96) contains eight spectrophotometers. White light is piped to a row of probes that move on a robotic arm. Fiber optic sensors are picked up by the probes and moved to a 96-well plate containing samples. One of the target molecules is immobilized on the biosensor surface. Then sensors are moved to wells containing the binding partner in solution. BLI monitors association of the binding partner with the immobilized molecule, and then monitors dissociation after moving the sensors to solution without the binding partner. Binding of molecules to the biosensor surface leads to changes in optical interference between light waves that reflect back to the spectrophotometers from an internal surface and from the external interface between sensor and solution. These changes in interference can be quantified and used to determine kinetic rates of binding and dissociation, as summarized in the animation of Figure 1.
We have applied BLI to measure interactions between the catalytic complex of bacterial ATP synthase and its &#949; subunit, which can auto-inhibit the enzyme. ATP synthase is a membrane-embedded rotary nanomotor that catalyzes synthesis and hydrolysis of ATP4.&#160;The catalytic complex (F1) can be isolated in a soluble form that works as an ATPase. Subunit &#949; has two domains: the N-terminal domain (NTD) is necessary for proper assembly and functional coupling of the enzyme but does not interact directly with the catalytic subunits; the C-terminal domain (CTD) can inhibit the enzyme by interacting with multiple catalytic subunits5,6. This &#949;-mediated regulation is specific to bacterial ATP synthases and is not observed in the mitochondrial homologue. ATP synthase has emerged as a target for antibacterial drugs, as shown by recent FDA approval of bedaquiline to treat drug-resistant tuberculosis7. Thus, targeting &#949;&#8217;s inhibitory role for drug discovery could yield antibacterials that do not inhibit the mitochondrial ATP synthase. With the isolated catalytic complex (F1), &#949; becomes a dissociable subunit. However, with &#949; bound to F1, the &#949;CTD can undergo a dramatic conformational change, partially inserting into the enzyme&#8217;s central cavity and forming an inhibitory state that is unlikely to dissociate directly6,8. We use BLI to measure kinetics of F1/&#949; binding and dissociation, and indirectly, to examine allosteric effects of catalytic-site ligands on &#949;&#8217;s conformation.
In our system, &#949; was chosen for immobilization on the sensor surface since BLI signal (like SPR) is sensitive to the mass of the molecules binding at the surface. The &#949; subunit is small (~15 kDa) relative to the main F1 complex (~347 kDa). Thus, a larger BLI signal will result from binding of F1 to immobilized &#949;. In order to monitor F1 dissociation, which can be very slow,&#160;&#949;&#160;must be strongly immobilized. Thus we chose to biotinylate&#160;and immobilize it on streptavidin-coated biosensors.&#160;Proteins can be biotinylated by (i) random modification of surface lysines9, (ii) reaction of a unique native or engineered cysteine with a biotin-maleimide reagent10 or (iii) genetically adding a specific biotin-acceptor peptide that is enzymatically biotinylated during in vivo expression of the tagged protein11. In our system, &#949; is biotinylated using method (iii)8. Once biotin-tagged &#949; is immobilized on streptavidin sensors, BLI can measure the binding and dissociation of F1 that has been depleted of subunit &#949; (F1(-&#949;)). For the experiments described here, preliminary assays had been done to determine reasonable amounts of the biotinylated protein to immobilize on the sensors. This can vary, depending on the molecular weight of the protein and its binding partner, but the goal is to determine a minimal amount of immobilized protein that provides (i) acceptable signal-to-noise for binding kinetics with a low concentration of the binding partner (below KD) and (ii) minimal distortion of binding kinetics with near-saturating concentration of the binding partner. Also, stoichiometry of biotinylation may vary (but avoid &#62;1 mol biotin/mol protein), so some initial assay may be needed for each new lot of biotinylated protein to confirm that a consistent BLI signal can be achieved during immobilization on the streptavidin-coated sensors.

<table><tbody><tr><td>Octet-RED96</td><td>Pall/Fort&amp;eacute;Bio</td><td>30-5048</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A6003-10G</td><td>Fatty Acid free</td></tr><tr><td>Biosensor/Streptavidin</td><td>Pall/Fort&amp;eacute;Bio</td><td>18-5019</td><td>Tray of 96 sensors</td></tr><tr><td>Microtiter plate</td><td>Greiner Bio-one</td><td>655209</td><td>Black, Polypropylene</td></tr><tr><td>Data Acquisition software</td><td>Pall/Fort&amp;eacute;Bio</td><td>Version 6.4</td><td>Newer versions available</td></tr><tr><td>Data Analysis software</td><td>Pall/Fort&amp;eacute;Bio</td><td>Version 6.4</td><td>Newer versions available</td></tr></tbody></table>

bio-layer interferometry for measuring kinetics of protein-protein interactions and allosteric ligand effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

Real time binding and dissociation BLI kinetics are shown in Figure 3. This experiment was done with assay buffer in association and dissociation. This experiment was started with a 10 min baseline step since the sensors had been prewet only briefly. Next, biotinylated &#949; was loaded on the sensors. No detectable dissociation of &#949; occurred throughout all remaining steps as seen from the reference curve (G) which had no binding partner added. A second reference sensor (H) was devoid of immobilized...

Watch this Scientific Journal Video about Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects at JoVE.com

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

bio-layer-interferometry-for-measuring-kinetics-protein-protein

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

29.0K Views.  SUNY Upstate Medical University. The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its ε subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with ε’s inhibitory C-terminal domain.

Video: Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and scalable technique efficiently functionalizes metallic nanoparticles with thiol-ligands in a miscible water/organic solvent mixture allowing for rapid grafting of thiol groups onto the gold nanoparticle surface. The hydrophobic ligands on the nanoparticles then quickly phase separate the nanoparticles from the aqueous based suspension and confine them to the air-fluid interface. This drives the ligand-capped nanoparticles to form monolayer domains at the air-fluid interface.&#160; The use of water-miscible organic solvents is important as it enables the transport of the nanoparticles from the interface onto template-free substrates.&#160; The flow is mediated by a surface tension gradient3,4 and creates macroscopic, high-density, monolayer nanoparticle-ligand films.&#160; This self-assembly technique may be generalized to include the use of particles of different compositions, size, and shape and may lead to an efficient assembly method to produce low-cost, macroscopic, high-density, monolayer nanoparticle films for wide-spread applications.

A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and ...

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

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.

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 ...

Biochemistry

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

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 ...

Quantitative Analysis of RPA-ssDNA Binding Kinetics Using Label-Free Methods

Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of various pathways. Key factors such as the structure, sequence, and length of a DNA molecule can significantly influence protein binding. Bio-layer interferometry (BLI) is a label-free technique that measures binding kinetics between molecules, offering a straightforward and precise approach to quantitatively study protein-DNA interactions. A major advantage of BLI over traditional gel-based methods is its ability to provide real-time data on binding kinetics, enabling accurate measurement of the equilibrium dissociation constant (KD) for dynamic protein-DNA interactions. This article presents a basic protocol for determining the KD value of the interaction between a DNA replication protein, replication protein A (RPA), and a single-stranded DNA (ssDNA) substrate. RPA binds ssDNA with high affinity but must also be easily displaced to facilitate subsequent protein interactions within biological pathways. In the described BLI assay, biotinylated ssDNA is immobilized on a streptavidin-coated biosensor. The binding kinetics (association and dissociation) of RPA to the biosensor-bound DNA are then measured. The resulting data are analyzed to derive precise values for the association rate constant (ka), dissociation rate constant (kd), and equilibrium binding constant (KD) using system software.

This article describes a protocol for studying DNA-protein interactions using a streptavidin-based biolayer interferometry (BLI) system. It outlines the essential steps and considerations for utilizing either basic or advanced binding kinetics to determine the equilibrium binding affinity (KD) of the interaction.

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of ...

Live Cell Single-Molecule Imaging-Based Quantification of Protein Interactions

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

Author Spotlight: Evaluation of Protein-Condensate Dynamics in Live Human Cells

Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells.

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number ...

Biology

Biolayer Interferometry Technology to Detect Interactions between Ligand and Analyte

Turn on the BLItz machine. Ensure that the machine is connected to the computer through a USB data output port at the back of the machine.
On the computer, open the associated software and click on &#34;Advanced Kinetics&#34; on the left-hand side of the screen. In the software, type out all appropriate information about the experiment under each respective heading. Click on &#34;Biosensor Type&#34; and choose &#34;Ni-NTA&#34; from the dropdown menu. The duration of each step can be changed from Default as needed. For optimal results, use a minimum of 30 seconds for Initial Baseline and Baseline and 120 seconds for Association and Dissociation.
Remove the hydrated Nickel-NTA-biosensor from the PCR tube, and affix it to the biosensor mount on the machine, by sliding the wide portion of the biosensor onto the mount.
Place a 0.5-milliliter black microcentrifuge tube into the tube holder of the machine and pipette 400 microlitres of BLI buffer into it. Close the cover of the machine such that the biosensor tip becomes submerged in the buffer in the microcentrifuge tube. Click &#34;Next&#34; on the software to begin recording the initial baseline.
After the initial baseline step has finished recording, open the cover of the machine. Move the slider to the right. Pipette 4 microlitres of a dialyzed His-tagged ligand onto the drop holder, and close the cover of the machine, which will automatically begin recording the loading step.
After the loading step has finished recording, open the cover of the machine. Move the slider to the left, such that the tube holder is once again situated in front of the black arrow. Close the lid of the machine, and ensure that the biosensor tip is submerged into the BLI buffer of the tube in the tube holder. The machine and software will automatically begin recording the baseline step.
After the baseline step has finished recording, open the cover of the machine. Remove the drop holder and clean it by pipetting out any protein and rinsing it with double-deionized water for a total of five times. Use a tissue wipe to clean the surface of the drop holder after the last wash. Replace the drop holder back onto the machine. Move the slider on the machine to the right such that the drop holder is once again situated in front of the black arrow.
Pipette 4 microlitres of a dialyzed analyte onto the drop holder and close the cover of the machine, which will automatically begin recording the association step. After the association step has finished recording, open the cover of the machine. Move the slider on the machine to the right such that the tube holder is once again situated in front of the black arrow, and close the cover of the machine, which will automatically begin recording the dissociation step.