Research at the Lewinson lab is supported by grants from the Israel Academy of Science, the Rappaport Institute for Biomedical research, the Meriuex Research Foundation, and the Marie Curie career reintegration grant (OL). EV is supported by the Fine scholarship and by the Technion Faculty of Medicine. NLL is supported by grants from the Israel Academy of Science.

1. Protein Sample and Buffer Preparation
<ol>
	<li>Sample preparation: purify the proteins of interest and make sure all aggregates are removed, by injecting the protein prior to the experiment onto a gel-filtration column or by ultracentrifugation (usually 10 min at 100,000 x g is sufficient). 
	NOTE: Although desirable, highly pure protein preparations are not a must. SPR has been successfully used with less-than-perfect purifications and even with total extract8,9.</li>
	<li>Buffer preparation:
	<ol>
		<li>Prepare the buffer of the experiment (termed &#8220;running buffer&#8221;, or RB). For the protocol described here, use 50 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.05% (w/v) DDM (n-Dodecyl &#946;-D-maltoside). Keep in mind that the choice of buffer composition can be readily modified according to the studied system.</li>
		<li>Filter all buffers and protein samples using 0.22 &#181;m filters. Make sure in advance that the filters are protein-compatible. In general, recently marketed SPR platforms contain an in-line de-gasser; if this is not the case, de-gas the buffers prior to use.</li>
		<li>Dialyze overnight or dilute the samples against the RB to avoid buffer mismatches. Dialysis (or any other method of buffer exchange) is especially advisable when using chemicals of high viscosity (e.g., sucrose, glycerol, DMSO). Before connecting the RB bottle to the pump inlet keep aside 10 ml of the RB in a separate tube.</li>
	</ol>
	</li>
	<li>Dilute the concentrated ligand stock into RB to a final concentration of ~20 &#181;g/ml. As a rule of thumb, for stable ligand immobilization, use low ligand concentrations with longer injections at low flow, as opposed to high concentrations, high flow and shorter injections.</li>
	<li>Dilute the analyte into the RB to the desired concentration. Typically, test the range of concentrations for a system of unknown affinity from 10 &#181;M to 10 nM in ten-fold dilutions. Always start with the lower concentrations first.</li>
</ol>
2. Sensor Chip
<ol>
	<li>Chip selection: Use a nitrilotriacetic acid (NTA) coupled chip suitable for capturing proteins with an oligo-histidine tag.</li>
	<li>Chip usage:
	<ol>
		<li>When using a new chip, take the chip out from the sheath, rinse gently with double distilled water (DDW), dry off carefully remaining liquids using delicate wipers, and make sure not to touch the gold layer surface. Place the chip back into its sheath in the correct orientation (the insertion is smooth when the chip is properly placed).</li>
		<li>When reusing a chip, take out the stored chip from the buffer, rinse thoroughly with DDW and dry gently as stated above, and place into its original sheath. 
		NOTE: The accumulation of non-specific junk material on the chip can affect its &#39;viability&#39;. This can be monitored by comparing the RUs prior to ligand immobilization and post stripping. Though many measurements can be done using the same NiNTA chip, the issue of its longevity is not a &#39;clear cut&#39; and varies greatly between different chips.</li>
	</ol>
	</li>
	<li>Chip docking: Open the sensor chip door and place the chip in its sheath. When using a new chip, input the chip&#39;s serial number to keep usage record. Close the door and press &#39;dock chip&#39;.</li>
</ol>
3. Temperature Settings
<ol>
	<li>Set the chip cell temperature to 25 &#176;C (or the preferred experiment temperature).</li>
	<li>Optional: set the samples compartment to 7 &#176;C to keep the proteins stable throughout the experiment.</li>
</ol>
4. Solutions for Loading and Stripping
Prepare the following solutions for loading and stripping: 0.5 mM NiCl2 in RB, 350 mM EDTA in RB (add detergent to EDTA solution to a final concentration as in RB), 0.25% SDS in H2O, and 100 mM HCl in H2O. When using micro-centrifuge tubes and not SPR designated tubes, do not forget to cut off the caps of the tubes.
5. Prime the SPR Instrument with RB
6. Start a New Run
<ol>
	<li>Set the flow rate that will be used during the experiment. To obtain the results described here set the flow to 50 &#181;l/min. 
	NOTE: This parameter can be modified during the experiment.</li>
	<li>Define the flow paths and reference subtraction. To obtain the results described here set the paths Fc=1 and Fc=2, subtraction Fc=2-1. 
	NOTE: The program will display in real-time the subtraction of two measured cells. Keep in mind that this parameter cannot be altered during the experiment.</li>
	<li>Select the suitable sample rack (it can influence the sample volume consumption).</li>
	<li>Save the results file.</li>
</ol>
7. Experiment Cycles
Each experiment is composed of cycles, keep a clear record of the injections done in each cycle, and separate the ligands&#8217; loading, the buffer&#39;s blank injection, and the analyte injection into different cycles.
<ol>
	<li>Cycle 1: Chip preparation and nickel loading
	<ol>
		<li>Make sure the flow and paths are set according to step 6.1 and 6.2.</li>
		<li>Inject 350 mM EDTA for 1 min to wash away leftovers.</li>
		<li>Set flow to 10 &#181;l/min.</li>
		<li>Inject 0.5 mM NiCl2 for 2 min. 
		NOTE: Now the chip resin is coated by nickel ions.</li>
	</ol>
	</li>
	<li>Cycle 2: Ligands immobilization
	<ol>
		<li>Set flow to 15 &#181;l/min, flow path Fc=2.</li>
		<li>Inject ligand A until reaching RU values that are 10-20 fold of its molecular weight. For example, load a ligand of 50 kDa to 500-1,000 RUs. Keep a record of the RU value achieved.</li>
		<li>Set flow to 15 &#181;l/min, flow path Fc=1.</li>
		<li>Inject ligand B (control) up to the same RU value as that of ligand A. Monitor the subtraction sensogram (Fc=2-1) on-line to help control the injection length.</li>
		<li>Set flow to 50 &#181;l/min, flow path Fc=1 and Fc=2, wash the system for 5-20 min until the baseline (Fc=2-1) stabilizes.</li>
	</ol>
	</li>
	<li>Cycle 3: Blank injection. This injection will serve for blank subtraction, therefore include all components that will be injected with the analyte, except the analyte itself. 
	NOTE: Injection length can vary depending on the time it takes to reach equilibrium (signal plateaus).
	<ol>
		<li>Set flow to 15 &#181;l/min, flow path Fc=1 and Fc=2, Fc=2-1 subtraction.</li>
		<li>Insert the &#39;wait&#39; command (referred hereafter as &#39;wait&#39;) for 30 sec (to have a stable baseline).</li>
		<li>Inject 15 sec RB. 
		NOTE: The duration of injection will vary between different systems, depending on their pre-equilibrium kinetic constants (mostly the ka).</li>
		<li>Wait 120-600 sec to record the dissociation phase. 
		NOTE: The length of this step varies according to the kd: The slower the dissociation the longer this step needs to be. For very slow dissociating complexes (kd &#60;10-4 sec-1), wait 10-15 min and then proceed to surface regeneration (see later).</li>
	</ol>
	</li>
	<li>Cycle 4: Analyte injection
	<ol>
		<li>Copy/paste the exact conditions used in the reference injection (cycle 3) so these two injections can be later subtracted. Change the location of the slot in the rack from the one holding the control solution to one that holds the analyte solution. Choose a concentration that is slightly above the expected KD. If no prior knowledge is available, choose 1 &#181;M as a good starting point.</li>
	</ol>
	</li>
	<li>Cycle 5: Buffer injection
	<ol>
		<li>Repeat cycle 3.</li>
	</ol>
	</li>
	<li>Cycle 6: Analyte injection
	<ol>
		<li>Repeat cycle 4.</li>
	</ol>
	</li>
	<li>Cycle 7: Chip strip off
	<ol>
		<li>Set flow to 50 &#181;l/min.</li>
		<li>Inject 350 mM EDTA for 1 min. Repeat this step two more times. Do not use a single injection of 3 min as this may damage the chip.</li>
		<li>Inject 0.25% SDS for 1 min. Repeat this step two more times. Do not use a single injection of 3 min as this may damage the chip.</li>
		<li>If baseline level is not reached, inject 100 mM HCl for 1 min as additional stripping step.</li>
		<li>Insert the &#39;end run&#39; command that switches to standby mode.</li>
	</ol>
	</li>
	<li>Chip storage
	<ol>
		<li>Undock the sensor chip and take it out of the instrument.</li>
		<li>Take the sensor out from its sheath, avoid touching the surface, rinse with DDW, and place in a 50 ml tube containing RB. Store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
8. Data Analysis Using Designated Evaluation Software
<ol>
	<li>Perform the following steps to process the raw data obtained by SPR before derivation of the kinetic parameters.
	<ol>
		<li>Using the evaluation software, open the results file, select cycles 3-6 Fc= 2-1 (reference subtracted data).</li>
		<li>Axis shift:
		<ol>
			<li>Display cycles 3-6.</li>
			<li>Zero the Y-axis value of the baseline (the initial 30 sec wait time in each cycle) to of all four curves.</li>
		</ol>
		</li>
		<li>Align the X- axis of the four curves. Choose pronounced features (e.g., spikes of injection start or finish) to perfectly align the four curves along the X-axis. Set the time of the start of analyte (or control buffer) to zero.</li>
	</ol>
	</li>
	<li>Reference subtraction
	<ol>
		<li>Subtract the background response by subtracting curve of cycle 3 from curve of cycle 4 and curve of cycle 5 from curve of cycle 6. Use the curve subtraction operation that can be found in the Y-axis shifts menu. 
		NOTE: It is important to perform this step as it annuls any nonspecific components (unrelated to the analyte) that may have influenced the surface refractive index.
		<ol>
			<li>Save the subtracted curves in the project&#8217;s sheet under a different name.</li>
		</ol>
		</li>
		<li>Refer to these curves as &#8220;doubly subtracted curves&#8221;: the first subtraction is the 2-1 subtraction performed on both injections, and the second is the subtraction of one curve from another. 
		NOTE: Such double referencing is the gold standard in SPR experiments.</li>
		<li>Overlay the subtracted curves by performing axis shift as explained before.</li>
	</ol>
	</li>
	<li>Determination of kinetics constants and model fitting
	<ol>
		<li>Conducting a kinetic experiment
		<ol>
			<li>Inject a series of 6-7 analyte concentrations to have a reliable of data for kinetic constants determination. Choose analyte concentrations from 10-fold above to 10-fold below the estimated KD, in 2-3-fold dilutions. Include the &#8220;zero&#8221; concentration, later used for double reference subtraction (see above). Conduct injections in triplicates and in random order.</li>
			<li>Align and subtract all curves with respect to the Y-axis and X-axis before attempting model fitting.</li>
		</ol>
		</li>
		<li>Model fitting
		<ol>
			<li>Select a suitable analysis program. 
			NOTE: Most available fitting programs offer several models that may be applied for fitting and determination of kinetic constants.</li>
			<li>Apply the simplest model (Langmuir) assuming a reversible 1:1 interaction between the analyte and the ligand for fitting. Unless convincing data is available from other experimental methods for the existence of a more complex interaction, always use the simple Langmuir model.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Rich, R. L., Myszka, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+surface+plasmon+resonance+biosensor+analysis%5BTitle%5D)+AND+%22Current+Opinion+in+Biotechnology%22%5BJournal%5D)">Advances in surface plasmon resonance biosensor analysis.</a> Current Opinion in Biotechnology. 11, 54-61 (2000).</li>
<li>Rich, R. L., Myszka, D. G. H. igher-throughput <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((label-free%2C+real-time+molecular+interaction+analysis%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">label-free, real-time molecular interaction analysis.</a> Analytical Biochemistry. 361, 1-6 (2007).</li>
<li>Helmerhorst, E., Chandler, D. J., Nussio, M., Mamotte, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+and+label-free+bio-sensing+of+molecular+interactions+by+surface+plasmon+resonance%3A+a+laboratory+medicine+perspective%5BTitle%5D)+AND+%22The+Clinical+Biochemist+Reviews%22%5BJournal%5D)">Real-time and label-free bio-sensing of molecular interactions by surface plasmon resonance: a laboratory medicine perspective.</a> The Clinical Biochemist Reviews. 33, 161(2012).</li>
<li>Kyo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+MafG+interaction+with+Maf+recognition+element+arrays+by+surface+plasmon+resonance+imaging+technique%5BTitle%5D)+AND+%22Genes+to+Cells%22%5BJournal%5D)">Evaluation of MafG interaction with Maf recognition element arrays by surface plasmon resonance imaging technique.</a> Genes to Cells. 9, 153-164 (2004).</li>
<li>Katsamba, P. S., Park, S., Laird-Offringa, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetic+studies+of+RNA%E2%80%93protein+interactions+using+surface+plasmon+resonance%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Kinetic studies of RNA–protein interactions using surface plasmon resonance.</a> Methods. 26, 95-104 (2002).</li>
<li>Vigonsky, E., Ovcharenko, E., Lewinson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+molybdate%2Ftungstate+ABC+transporters+that+interact+very+differently+with+their+substrate+binding+proteins%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U+S+A%22%5BJournal%5D)">Two molybdate/tungstate ABC transporters that interact very differently with their substrate binding proteins.</a> Proc. Natl. Acad. Sci. U S A. 110, 5440-5445 (2013).</li>
<li>Lewinson, O., Lee, A. T., Locher, K. P., Rees, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+distinct+mechanism+for+the+ABC+transporter+BtuCD-BtuF+revealed+by+the+dynamics+of+complex+formation%5BTitle%5D)+AND+%22Nat.+Struct.+Mol.+Biol%22%5BJournal%5D)">A distinct mechanism for the ABC transporter BtuCD-BtuF revealed by the dynamics of complex formation.</a> Nat. Struct. Mol. Biol. 17, 332-338 (2010).</li>
<li>Kyo, M., Usui-Aoki, K., Koga, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-Free+Detection+of+Proteins+in+Crude+Cell+Lysate+with+Antibody+Arrays+by+a+Surface+Plasmon+Resonance+Imaging+Technique%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Label-Free Detection of Proteins in Crude Cell Lysate with Antibody Arrays by a Surface Plasmon Resonance Imaging Technique.</a> Analytical Chemistry. 77, 7115-7121 (2005).</li>
<li>Mori, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+protein+kinase+activities+of+cell+lysates+using+peptide+microarrays+based+on+surface+plasmon+resonance+imaging%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Evaluation of protein kinase activities of cell lysates using peptide microarrays based on surface plasmon resonance imaging.</a> Analytical Biochemistry. 375, 223-231 (2008).</li>
<li>Mol, N., Fischer, M. E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMol%2C+N+%22Surface+Plasmon+Resonance+Vol.+627+Methods+in+Molecular+Biology%22">Surface Plasmon Resonance Vol. 627 Methods in Molecular Biology</a>. Mol, N. J., Fischer, M. J. E. 1, Humana Press. 1-14 (2010).</li>
<li>Van Der Merwe, P. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aVan+Der+Merwe%2C+PA+%22Surface+plasmon+resonance.+Protein%E2%80%93+ligand+interactions%3A+hydrodynamics+and+calorimetry%22">Surface plasmon resonance. Protein– ligand interactions: hydrodynamics and calorimetry</a>. , Oxford University Press. Oxford. 137-170 (2001).</li>
<li>Lewinson, O., Lee, A. T., Locher, K. P., Rees, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+distinct+mechanism+for+the+ABC+transporter+BtuCD-BtuF+revealed+by+the+dynamics+of+complex+formation%5BTitle%5D)+AND+%22Nat.+Struct.+Mol.+Biol%22%5BJournal%5D)">A distinct mechanism for the ABC transporter BtuCD-BtuF revealed by the dynamics of complex formation.</a> Nat. Struct. Mol. Biol. 17, 332-338 (2010).</li>
</ol>

All authors declare that they have no competing financial interests.

SPR is a highly sensitive method to study molecular interactions, and is often the only approach that provides real-time monitoring of transient (yet important) interactions. An example is the transient interaction presented herein, that could not be detected by any other method (pull-down assays, liposomes sedimentation assays6). Moreover, while other methods are limited to equilibrium measurements (whether quantitative or qualitative), SPR is one of the only techniques that measure also the pre-equilibrium kinetics.
Its heightened sensitivity is also its caveat. False-positive sensograms are readily recorded. It is therefore recommended to establish the existence of the interaction using other methods before attempting SPR measurements. Prior knowledge of the system&#8217;s characteristics (rough assessment of the KD, stable vs. transient interaction) is very helpful in setting up SPR experiments and evaluating the functional relevance of the obtained measurements. An important issue to keep in mind before starting SPR measurements is the quality of the protein samples. SPR is highly sensitive to aggregates and these must be removed from both the ligand and the analyte preparations (by gel filtration chromatography or ultracentrifugation when possible). Another source of noise and false signals may stem from buffer mismatches between the RB and the buffer of the analyte. This may be solved by dialyzing the analyte against the RB, or by using a highly concentrated preparation of the analyte which is diluted many folds (~100-1,000-fold) into the RB. False positive data can also result from non-specific interactions of the analyte with the chip&#39;s matrix or with the negative control. These interactions are of low affinity, so sometimes can be avoided by using low analyte concentrations. In addition, inclusion of low BSA and/or detergent concentrations (0.1 mg/ml and/or 0.05-0.1% (w/v), respectively) to the RB and to the injected analyte sample can decrease nonspecific interactions. Changing the content in the negative control cell (different negative control) is another approach for dealing with nonspecific binding issues. Alternatively, using another type of chip can also affect the quality of data. Various kinds of sensor chips are commercially available, based on different chemistries. In the most commonly used sensor chip (the CM-5 chip) carboxymethylated dextran is covalently attached to a gold surface. Proteins can be covalently coupled to the sensor surface using a variety of very simple surface chemistries (most commonly amine or thiol coupling). However, in our hands, when working with membrane proteins covalent immobilization often yields artifacts and is thus not the method of choice. In the context of studying a membrane protein, it is noteworthy that there are commercial chips that are coated with lipophilic molecules. These enable the immobilization of membrane proteins onto a native-like environment. Similar sensor chips can also be prepared &#39;in house&#39;. For a description of other coupling methods see10,11. The protocol described herein is based on the use of Ni-NTA chip for the immobilization of a His tagged, detergent solubilized transporter (the analyte is not His-tagged to avoid analyte binding to the chip surface). In our experience 6,12, kinetic constants derived from experiments conducted with Ni-NTA chips are in good agreement with those determined in native-like environments. Although Ni-NTA chips are relatively expensive, their primary advantage is that they can be stripped of ligand and re-used. Hundreds of measurements can be done per chip. Moreover, the ligand&#39;s orientation on the chip is determined by the position of the his-tag and is thus quite homogeneous.
Once an interaction has been successfully recorded by SPR, a series of controls and repeats are needed to verify the validity of the SPR signal. Verification of the following parameters may assist to validate the SRP data:
1. Specificity of the SPR signal: due to the high sensitivity of SPR, the use of appropriate negative controls is of special importance. Non-specific interaction recorded by SRP can be due to the adsorption of an analyte to the matrix. Therefore, an empty flow cell is often not the best option since a flow cell loaded with protein (the ligand) is not chemically equivalent to an empty flow cell. A better option is to use an inactive mutant variant of the ligand, one that is known not to bind the analyte. Alternatively, one can use a similarly mutated analyte. If such mutants are not available a homologous protein (but of different binding specificity) can be used 12. Examples of good negative controls are a homologous ligand (as described here), or a point mutation in one of the interacting partners that is known to abolish the association. Another recommended negative control is alteration of a post-translational modification (e.g., phosphorylation, methylation, thiol derivation) that is essential to the interaction. For example, de-phosphorylation of a tyrosine that is essential for SH2 domain interaction. Other negative controls may be system specific. When using SPR, the importance of using strict negative controls cannot be overstated.
2. Reproducibility of the SPR signal: SPR is an extremely reproducible method. When equal ligand amounts are loaded onto the biosensor chip and equal concentrations of the analyte are injected the repeats should perfectly align (see Figure 3).
3. Regeneration of the system: in interactions with a slow dissociation rate (kd), the analyte must be stripped from the ligand while preserving the functional and structural integrity of the latter. If dissociation and/or regeneration are incomplete, the SPR signal will gradually decay upon subsequent injections of the analyte. Typical regenerating conditions that can be tested are high concentrations of Mg2+ (up to 2 M), acidic or basic conditions (e.g., 10 mM glycine pH 2.5, 10 mM NaOH), high salt (up to 4 M NaCl), or high detergent concentration (e.g., 1% DDM). However, the regenerating effectiveness of these reagents (and others) is highly system-specific and thus regenerations conditions must be tested. In some cases, the interaction is so stable and strong that regeneration is never complete, as in the case of the E. coli vitamin B12 transport system (BtuCD-F) 7. Under such conditions, the only option is to strip the ligand from the biosensor chip and reload a fresh batch for each injection. In cases where the dissociation of the analyte from the ligand is fast and complete surface regeneration is unnecessary (see protocol and Figures 2-3).
4. Membrane proteins are always purified in the presence of detergent, which can affect the SPR sensitivity and reaction rates. However, in our experience with BtuCD, the measured rates are very similar. One always needs to obtain supporting evidence using complimentary approaches such as size exclusion chromatography (SEC), pull-down assays, liposome sedimentation assay, etc. For example a correlation between SPR and other techniques has been shown for the methionine, vitamin B12, and two molybdate systems 6,12.
Erroneous determination of the kinetic rate constants often arises during the fitting process. As a rule of thumb, always apply the simple Langmuir 1:1 interaction model for analysis kinetic experiments. The more complex models make additional assumptions of conformational changes, bivalent analyte, or sample heterogeneity. These assumptions should be made only if supported by evidence from other experimental approaches. Note that applying these models will most likely result in better fitting and lower Chi2 values, but this should not be viewed as a testimony for their reliability. The higher degrees of freedom afforded by the more complex models are usually responsible for the better fitting, rather than their mechanistic relevance.
One of the advantages of SPR is its broad chemical compatibility, especially with respect to detergents that are used to solubilize and purify membrane proteins. However, sucrose and glycerol (and other viscous solutions) that are often added during the purification process of membrane proteins have dramatic effects on the RU. It is therefore recommended to avoid these if possible, or at least reduce their concentration. When using viscous solutions, avoiding buffer mismatch becomes essential. Adding lipids to a detergent containing RB can amplify the binding signal by as much as ten-fold. However, such an endeavor is expensive (for most lipids) and tends to shorten the life span of the biosensor chips.
The tips and troubleshooting listed above may help while calibrating a new system. However, a given system often requires specific tweaking (e.g., choice of detergent, salt, pH) to achieve optimal results.
SPR has long been recognized as a powerful and unique approach to measure molecular interactions. In recent years the availability of various SPR platforms and their declining prices has made SPR more approachable. It is rapidly becoming the gold standard approach to study protein-protein interactions, target protein-drug interactions, and in discovering lead small molecule inhibitors.

Protein-protein interactions (PPI); the formation and dissociation of protein complexes, are key events in many biological processes (e.g., replication, transcription, translation, signaling, cell-cell communication). Semi-quantitative studies of PPI are often performed using pull-down or immuno-precipitation experiments. However, these (and similar) techniques are limited in the range of affinities that can be measured (low micromolar and higher affinity) due to the washing steps that are inherent to such techniques. Such &#8220;end-point&#8221; techniques cannot identify transient or low affinity interactions that are often of great biological consequences. In addition, the temporal resolution of such approaches is extremely limited, usually orders of magnitude slower than the rates of the reaction. SPR overcomes these shortcomings due to its heightened sensitivity and superior temporal resolution1,2. SPR is a label-free method, and as such molecular interaction can be measured (as long as the mass changes can be detected). In addition to PPI, it has been extensively used to characterize protein-ligand, protein-drug (or small molecule), protein-DNA, and protein-RNA interactions 3-5. Results are recorded and plotted in real time, which enables rapid modification of experimental conditions and flexible experimental design.
The physical principle behind SPR based instruments is the utilization of an optical system that measures a change of the refractive index on the sensor surface upon mass changes 2. One of the interacting partners (hereafter ligand) is immobilized onto a polymer matrix chip and the second molecule (hereafter analyte) is flowed over the surface. Analyte binding to the ligand alters the mass on the chip surface. This mass change is directly and proportionally related to changes in the refractive index of the surface. The results are plotted in real time and presented as response units (RU) as a function of time. Such a plot is termed a sensogram (e.g., Figure 1). Since SPR follows the complete time course of the interaction, pre-equilibrium kinetic rate constants are derived, in addition to equilibrium affinities. As detailed below, the pre-equilibrium behavior of a given system holds much information, and provides a very different perspective than equilibrium alone. Once the system is calibrated, experiments are very fast and require only small amounts of protein material (microgram to nanogram amounts). Collecting the complete kinetic information of a given system can be achieved in days. The high sensitivity of SPR affords measurements that are not possible using any other technique 6. However, this high sensitivity is a &#39;two-edged sword&#39; since it can be a major source for false positive data. Any factor that affects the reflective index is recorded and plotted on the sensogram. As such, appropriate controls must be used to eliminate false-positives, and support from complementary approaches is highly advised.
Figure 1 illustrates the progression of an SPR experiment using an NTA-coated sensor chip. The sensogram in panel A shows the injection of the nickel ions over the NTA matrix (unsubtracted data), and panels B-D display the data after subtracting the signal derived from the negative control cell. Red and blue arrows show injection start and end, respectively. Immobilization of the ligand onto the chip gradually alters the mass until injection is terminated. The stable plateau observed after termination of ligand&#39;s injection reflects the stable association of the ligand to the surface (Figure 1B, cycle 2). Once a stable baseline is achieved a buffer is injected over the ligand and the reference cell (Figure 1B, cycle 3).This injection serves as a &#39;blank control&#8221;, and will be subtracted during analysis. Upon injection, minor changes are recorded, reflecting a flow of mass through the chip. Then in a separate cycle (Figure 1B, cycle 4), the analyte is injected; the gradual increase in RU represents the analyte&#39;s association to the ligand. The binding sites become gradually occupied until equilibrium is reached. As soon as injection ends, a decline in RUs reflects the complex&#39;s dissociation. From such sensograms pre-equilibrium and equilibrium rate constants can be derived (see later). Figure 1 depicts a transient interaction between the ligand and analyte. When the interaction is more stable, the decline in the RU level is slower, reflecting a lower kd.
Herein, we describe an SPR experiment aimed at characterizing the interaction between a membrane transporter (detergent solubilized) and its functional partner, its cognate substrate binding protein 6,7. The chosen model system is an ATP binding cassette (ABC) transporter, ModBC-A of Archeaglobus fulgidus. This system was selected for its highly reproducible results, high signal to noise ratio, and classical &#39;on/off&#39; rates. Additionally, homologues ABC transporters are available to serve as important negative controls. The transporter, ModBC (ligand A) is extracted from the membrane using detergent, purified and immobilized onto the chip. Its soluble interacting partner, ModA, is the analyte. As a negative control ligand, a different ABC transporter RbsBC is used (&#8220;ligand B&#8221;).

<table><tbody><tr><td>Biacore T200</td><td>GE Healthcare</td><td>28-9750-01</td><td></td></tr><tr><td>Sensor Chip NTA&amp;nbsp;</td><td>GE Healthcare</td><td>14100532</td><td></td></tr><tr><td>BIAevaluation</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Biacore T200 Software&amp;nbsp;</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Nickel chloride</td><td>Sigma</td><td>N6136</td><td></td></tr><tr><td>Sodium tungstate dihydrate</td><td>Sigma</td><td>T2629</td><td></td></tr><tr><td>Sodium Chloride&amp;nbsp;</td><td>Bio-Lab LTD.</td><td>19030591</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>Ethyldiamine-tetraacetic acid disodium salt dihydrate (EDTA)</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>n-Dodecyl-&amp;beta;-D-Maltopyranoside (DDM)</td><td>Affymetrix</td><td>D310</td><td></td></tr><tr><td>Sodium dodecyl sulfate solution&amp;nbsp;</td><td>Sigma</td><td>05030</td><td></td></tr><tr><td>Kimwipes KIMTECH</td><td>Kimberly-Clark</td><td>34120</td><td></td></tr><tr><td>Hydrochloric acid</td><td>GADOT</td><td>7647-01-0</td><td></td></tr><tr><td>Nylon (NY) Membrane Filter</td><td>Sartorius</td><td>25007--47------N</td><td></td></tr><tr><td>ModBC ABC transporter</td><td>Lewinson lab</td><td></td><td></td></tr><tr><td>RbsBC ABC transporter</td><td>Lewinson lab</td><td></td><td></td></tr><tr><td>ModA Substrate Binding Protein</td><td>Lewinson lab</td><td></td><td></td></tr></tbody></table>

real time measurements of membrane protein:receptor interactions using surface plasmon resonance (spr)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

In the system described herein, a NiNTA chip is used to immobilize the His-tagged membrane transporter 6,7. Being a homo-dimer, each transporter is doubly tagged, improving its binding to the NiNTA chip. Following nickel loading, ligand A (the transporter of interest) is immobilized onto Fc=2, up to ~3,500 RU (protocol cycle 2, Figure 2A black label). Then, using the same flow and injection duration, ligand B (the control ligand) is injected onto Fc=1, initially reaching only up to a ~3,000 RU. To make sure that similar protein amounts are immobilized on both flow cells; ligand B is further loaded, using shorter injection, while carefully monitoring the loading up to 3,500 RUs. The &#39;stairs&#39; shape represents the gradual increase in mass on Fc=1 upon each injection (Figure 2A gray label). An on-line monitoring of Fc=2-1 helps controlling the equal ligand loading. Figure 2B demonstrates the Fc=2-1 subtraction, i.e., subtracting the maximum RU value in Fc=2, from the step-wise increments in the RU value in Fc=1. It is important to vary the duration of the ligands&#8217; injections to achieve equal loading of the investigated and control ligands. In contrast, when injecting the analyte, the duration of the injection must be kept constant for all concentrations used. Figure 3A and 3B demonstrate the responses measured by injecting a &#39;blank analyte&#39; (i.e., RB omitting only the analyte) in cycles 3 and 5, and the analyte injections (in cycles 4 and 6). Note that both sensograms represent the Fc=2-1 subtraction. In each of the cycles (3-6) an identical analyte injection (blank or a given concentration) is applied onto the two flow cells, immobilized with different ligands. The two repeats superimpose very well, demonstrating the high reproducibility of SPR. A response is recorded in both cases; ~3 RU for the blank versus ~40 RU for the analyte. These values represent a signal to noise ratio of ~13. To obtain the double-referenced sensograms, representing only the specific binding response, the blank injection is now subtracted from the analyte injection (~37 RU, Figure 3C). The sensogram recorded reveals the characteristic pattern of a transient interaction. Upon injection, the association phase is characterized by a rapid increase in the amount of ligand-bound analyte, reaching steady state after ~7 seconds. The steady state is maintained for as long as the analyte is injected. When the injection terminates, the cells are washed with RB triggering the dissociation phase. The complex rapidly dissociates, and as the analyte is washed away from the ligand the SPR signal returns to baseline.
To gain quantitative data (pre-equilibrium and equilibrium constants), a range of analyte concentrations is injected in duplicates or triplicates (see 8.3.1 in protocol). Figure 4 shows injections of different analyte concentrations (duplicates per each concentration), keeping a constant injection length. The different analyte concentrations are injected in a random order, and the double blanked sensograms are X-axis aligned prior to analysis. To derive the affinity and rate constants, a model fitting is applied (see section 8.3.2.2 in protocol). The calculated equilibrium dissociation constant (KD) for the ModBC-A interaction shown in Figure 4 is ~3 x 10-6 M, with a moderately fast ka (~104 M-1 sec-1) and fast kd (~0.1 sec-1).
<img alt="Figure 1" src="/files/ftp_upload/51937/51937fig1highres.jpg" /> 
Figure 1. Illustration of the steps of an SPR experiment using a Ni-NTA sensor chip. (A) Injection of the nickel over the NTA matrix on flow cells Fc=1 and Fc=2 (shown are unsubtracted curves). The increase in mass is low (~30-50 RUs). (B-D) Illustration of the Fc=2-1 sensogram recorded during an SPR experiment. In cycle 2 as the his-tagged ligand is injected over the Ni-charged surface, mass gradually accumulates. Injection is terminated when reaching a final RU value ranging between 1,000-5,000 RU (depending on the ligand&#39;s MW). A baseline is formed, reflecting the stable binding of the ligand to the chip. In cycle 3, a blank injection is preformed, injecting the components of analyte sample excluding only the analyte. In this injection often a low response is recorded (1-5 RUs). In cycle 4 the analyte is injected using the same regime as the blank injection. The increasing RUs reflect the mass change on the surface, i.e., the binding of the analyte to the ligand. The signal rapidly increases and then plateaus as the system reaches equilibrium. When the injection ends, the analyte dissociates from the ligand, resulting in a decrease in the signal and return to baseline levels.
<img alt="Figure 2" src="/files/ftp_upload/51937/51937fig2highres.jpg" /> 
Figure 2. On line monitoring of ligand loadings. (A) Unsubtracted curves of ligand loading. The ligand of interest (ligand &#8220;A&#8221;) is loaded to flow cell 2 (Fc=2, black) until reaching the desired RU level (~3,500 RU in this example). Then, the negative control ligand (ligand &#8220;B&#8221;) is step-wise loaded onto flow cell 1 (Fc=1, gray) until an identical RU level is reached. (B) The two injections performed in (A) monitored in real time as an Fc=2-1 subtraction. This monitoring facilitates identical loading of ligands of interest and control.
<img alt="Figure 3" src="/files/ftp_upload/51937/51937fig3highres.jpg" /> 
Figure 3. Double referencing. (A) Duplicates of a 2-1 subtraction (i.e., Fc=2-1) of the blank injection. (B) As in (A), only analyte is injected. (C) Final sensograms after subtraction of the background response shown in (A) from the measured response in (B). These curves are referred to as &#39;double blanked&#8221; or &#39;double referenced&#8221;. Duplicates are of sequential cycles.
<img alt="Figure 4" src="/files/ftp_upload/51937/51937fig4highres.jpg" /> 
Figure 4. Determination of kinetic rate constants by injections of multiple analyte concentrations (in duplicates). The black lines are the fits that were calculated using a simple 1:1 Langmuir interaction model.

Watch this Scientific Journal Video about Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR at JoVE.com

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

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22.7K Views.  The Technion-Israel Institute of Technology.  Surface Plasmon Resonance (SPR) is een label vrij werkwijze voor het detecteren van biomoleculaire interacties in real time. Hierin, een protocol voor een membraaneiwit: wordt receptor interactie experiment beschreven, terwijl de discussie over de voors en tegens van de techniek. 

Video: Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

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 ...

Biochemistry

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Bioengineering

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Real-Time Detection of Dynamic Affinity between Biomolecules Using Surface Plasmon Resonance (SPR) Technology

Surface plasmon resonance (SPR) technology is a sensitive precise method for detecting viruses, pathogenic molecular proteins, and receptors, determining blood types, and detecting food adulteration, among other biomolecular detections. This technology allows for the rapid identification of potential binding between biomolecules, facilitating fast and user-friendly, non-invasive screening of various indicators without the need for labeling. Additionally, SPR technology facilitates real-time detection for high-throughput drug screening. In this program, the application field and basic principles of SPR technology are briefly introduced. The operation process is outlined in detail, starting with instrument calibration and basic system operation, followed by ligand capture and multi-cycle analysis of the analyte. The real-time curve and experimental results of binding quercetin and calycosin to KCNJ2 protein were elaborated upon. Overall, SPR technology provides a highly specific, simple, sensitive, and rapid method for drug screening, real-time detection of related pharmacokinetics, virus detection, and environmental and food safety identification.

Real-Time Detection of Dynamic Affinity between Biomolecules Using Surface Plasmon Resonance SPR Technology

The present study aims to elucidate the principle and methodology of surface plasmon resonance (SPR) technology, which finds versatile applications across multiple domains. This article describes SPR technology, its operational simplicity, and its remarkable efficacy, with the goal of fostering broader awareness and adoption of this technology among readers.

Surface plasmon resonance (SPR) technology is a sensitive precise method for detecting viruses, pathogenic molecular proteins, and receptors, determining ...

Medicine

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Surface Plasmon Resonance (SPR)

Surface plasmon resonance (SPR) is the underlying optical phenomenon behind label-free biosensors to evaluate the molecular affinity, kinetics, specificity, and concentration of biomolecules. In SPR, biomolecular interactions occur on a biosensor made of a thin layer of metal on a prism. Real-time interactions of biomolecules can be monitored by measuring the changes of light reflected off the underside of the metal.
This video describes the basic concepts of SPR and how it is used to analyze and visualize biomolecular interactions. This is followed by a sample preparation and experimental protocol for investigating binding rates using SPR. In the applications section, SPR imaging, localized SPR, and quantum dot enhanced SPR are explored.
<div class="chapter_text" id="script_0">
Surface plasmon resonance, or SPR, is the underlying phenomenon behind certain label-free biosensors for evaluating binding and adsorption interactions of biomolecules. Binding assays that require labeling, such as ELISA, can be a time-consuming process, and may alter the functionality of the analyte. In SPR, biomolecular interactions occur on a special sensor made of a thin layer of metal on one face of a prism. By monitoring the changes in light reflected off of the underside of the metal, SPR instruments visualize these interactions in real-time without the use of labels. This video will introduce the principles of SPR, a general procedure for SPR imaging, and some applications of in biochemistry.
</div>
<div class="chapter_text" id="script_1">
An SPR sensor is usually made of a thin layer of a noble metal atop the face of a prism. To take readings from the sensor, light is reflected off of the prism-metal interface into a photodetector. The reflected light will have a high intensity except at a certain angle, related to the electronic properties of the metal surface, known as the &#34;surface plasmon resonance angle&#34;.
As molecules bind to the surface, the electronic properties of the metal change, which in turn adjusts the angle. As new proteins attach, forming complexes, the angle will shift further. By measuring relative changes in the SPR angle, interactions like these can be monitored in real-time.
Another technique called localized, or &#34;L&#34;SPR, uses metal nanoparticles as the sensor surface. The properties that affect the SPR angle are highly localized to each nanoparticle, which improves sensitivity and signal resolution.
When investigating binding interactions with standard SPR, the sensor is generally mounted in a platform that becomes the floor of a flow cell in the instrument. The biomolecules of interest are carried through the flow cell by buffer solution. The sensor surface is often first coated with a substrate that has a high affinity for the metal. This ensures that a significant amount of ligand, which in turn binds to the analyte of interest, will be immobilized onto the sensor and reduces the likelihood that the ligand will dissociate during the procedure.
Once the ligand is immobilized on the sensor, the analyte is flowed over the sensor in buffer. By monitoring the change in the SPR angle over time as the analyte binds to the ligand, the binding rate and other kinetic information can be calculated.
The reflectance data can also be used for SPR imaging, or SPRi, by directing the reflected light to a CCD detector. This produces a high-contrast, high-resolution image of the entire sensor surface. Using SPR and the related techniques, questions can be answered about molecular affinity, kinetics, specificity, and concentration.
Now that you understand what is being measured in an SPR experiment, let&#39;s look at a procedure for investigating binding rates.
</div>
<div class="chapter_text" id="script_2">
Before beginning the procedure, the running and sample buffers must be prepared. The running buffer is used to deposit the ligand onto the sensor, and the sample buffer is used to deposit the analyte. The sensor chip is carefully cleaned and loaded into a sheath. Then, the device is placed into the instrument, where it becomes the bottom of the flow cell. The instrument software is set up for the experiment and subsequent analysis. If necessary, the sensor surface is primed with a substrate to capture the ligand. The ligand is flowed over the sensor surface in the running buffer, where it is captured by the substrate on the sensor surface.
Then, the analyte in the sample buffer is run through the flow cell, where it selectively binds to the immobilized ligand. The change in reflectance is plotted and compared against controls to determine rate constants and other reaction kinetics data for the investigated reaction.
</div>
<div class="chapter_text" id="script_3">
Now that you understand how an SPR experiment is performed, let&#39;s look at a few other applications of SPR in biochemistry.
Here, SPR imaging was used to evaluate proteins with an array of eleven receptors on a sensor. 3D graphs of reflectivity versus time and receptor concentration were prepared from the reflectivity data for each protein. These &#34;profiles&#34; are characteristic to each protein, and thus could subsequently be used for protein identification.
In this experiment, cell secretions were studied using a custom-made LSPR sensor. The sensor was also compatible with SPRi and fluorescence microscopy. Upon depositing the cell on the sensor, the interaction of cell secretions with the nanoparticle array could be measured with high spatial resolution.
Here, the use of quantum dots, nanoscale semiconductors, as an SPR signal enhancement agent mixed with the analyte was investigated. This enhanced &#34;nano-SPRi&#34; method was compared to assays by standard SPRi and the ELISA method. The nano-SPRi method significantly improved the sensitivity and limit of detection while still being less time-consuming than the ELISA method.
</div>
<div class="chapter_text" id="script_4">
You&#39;ve just watched JoVE&#39;s video on surface plasmon resonance. This phenomenon is used to monitor and image biomolecular interactions without the use of labels. This video introduced the principles of SPR, a typical protocol for performing an SPR experiment, and a few applications of SPR in biochemistry.
Thanks for watching!
</div>

Surface Plasmon Resonance SPR: Label-Free Detection

Surface plasmon resonance (SPR) is the underlying optical phenomenon behind label-free biosensors to evaluate the molecular affinity, kinetics, ...

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Surface Plasmon Resonance to Study Biomolecular Interactions Using a Sensor Chip

Source: Maier, I. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63541">Engineering Antiviral Agents via Surface Plasmon Resonance.</a> J. Vis. Exp. (2022)
This video describes the surface plasmon resonance, or SPR, technique for studying the interaction between two molecules using a sensor chip. The change in resonance angle and the intensity of the reflected light are detected as a function of the association and dissociation of two molecules, and this provides information about the interactions between the two molecules.

Source: Maier, I. Engineering Antiviral Agents via Surface Plasmon Resonance. J. Vis. Exp. (2022)
This video describes the surface plasmon resonance, or ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

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