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/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 ...

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JoVE Journal

Biology

22.6K 次观看.  The Technion-Israel Institute of Technology. 表面等离子共振（SPR）是一种无标记检测方法，实时生物分子相互作用。在这里，协议的膜蛋白：受体相互作用实验说明，在讨论该技术的优点和缺点。 

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

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

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 ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster ...