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>...

<ol>
	<li>Rich, R. L., Myszka, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+surface+plasmon+resonance+biosensor+analysis.">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. i. g. h. e. r. -. t. h. r. o. u. g. h. p. u. t. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=label-free,+real-time+molecular+interaction+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+and+label-free+bio-sensing+of+molecular+interactions+by+surface+plasmon+resonance:+a+laboratory+medicine+perspective.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+MafG+interaction+with+Maf+recognition+element+arrays+by+surface+plasmon+resonance+imaging+technique.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+studies+of+RNA&#8211;protein+interactions+using+surface+plasmon+resonance.">Kinetic studies of RNA&#8211;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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+molybdate/tungstate+ABC+transporters+that+interact+very+differently+with+their+substrate+binding+proteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+distinct+mechanism+for+the+ABC+transporter+BtuCD-BtuF+revealed+by+the+dynamics+of+complex+formation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+Detection+of+Proteins+in+Crude+Cell+Lysate+with+Antibody+Arrays+by+a+Surface+Plasmon+Resonance+Imaging+Technique.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+protein+kinase+activities+of+cell+lysates+using+peptide+microarrays+based+on+surface+plasmon+resonance+imaging.">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., Mol, N. J., Fischer, M. J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Surface Plasmon Resonance Vol. 627 Methods in Molecular Biology. 1, 1-14 (2010).</li><li>Van Der Merwe, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Surface plasmon resonance. Protein&#8211; ligand interactions: hydrodynamics and calorimetry. , 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+distinct+mechanism+for+the+ABC+transporter+BtuCD-BtuF+revealed+by+the+dynamics+of+complex+formation.">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 k...

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 border="0" cellpadding="0" cellspacing="0" width="601">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Name of Material/ Equipment</td>
			<td style="width:107px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biacore T200</td>
			<td style="width:107px;">GE Healthcare</td>
			<td style="width:119px;">28-9750-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Sensor Chip NTA&#160;</td>
			<td style="width:107px;">GE Healthcare</td>
			<td style="width:119px;">14100532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">BIAevaluation</td>
			<td style="width:107px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Biacore T200 Software&#160;</td>
			<td style="width:107px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Nickel chloride</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">N6136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Sodium tungstate dihydrate</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">T2629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Sodium Chloride&#160;</td>
			<td style="width:107px;">Bio-Lab LTD.</td>
			<td style="width:119px;">19030591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Trizma base</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:209px;">Ethyldiamine-tetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">E5134</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:209px;">n-Dodecyl-&#946;-D-Maltopyranoside (DDM)</td>
			<td style="width:107px;">Affymetrix</td>
			<td style="width:119px;">D310</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Sodium dodecyl sulfate solution&#160;</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">05030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Kimwipes KIMTECH</td>
			<td style="width:107px;">Kimberly-Clark</td>
			<td style="width:119px;">34120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Hydrochloric acid</td>
			<td style="width:107px;">GADOT</td>
			<td style="width:119px;">7647-01-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nylon (NY) Membrane Filter</td>
			<td style="width:107px;">Sartorius</td>
			<td style="width:119px;">25007--47------N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">ModBC ABC transporter</td>
			<td style="width:107px;">Lewinson lab</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">RbsBC ABC transporter</td>
			<td style="width:107px;">Lewinson lab</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">ModA Substrate Binding Protein</td>
			<td style="width:107px;">Lewinson lab</td>
			<td style="width:119px;"></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...

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 ...

real-time-measurements-membrane-proteinreceptor-interactions-using

Research

JoVE Journal

Biology

22.4K Views.  The Technion-Israel Institute of Technology. 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.