Name: labview-operated novel nanoliter osmometer for ice binding protein investigations
Uploaded: 2013-02-04T09:50:00
Duration: 9 min 32 s
Description: Watch this Scientific Journal Video about LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations at JoVE.com

This research was supported by the ISF, NSF, and ERC. We would like to acknowledge technical help with the temperature stage from Randy Milford, Michael Koren, Doug Shafer, and Jeremy Dennison. Assistance with software development was provided by Or Chen, Di Xu, Rajesh Sannareddy, and Sumit Bhattachary. We would like to thank our collaborators Prof. Peter L. Davies and Dr. Laurie A. Graham for the MpIBP protein and helpful discussions. We also thank lab members Dr. Maya Bar-Dolev, Yangzhong Qin, Dr. Yeliz Celik, Dr. Natalya Pertaya, Ortal Mizrahy, and Shlomit Guy for their user feedback.

0. Preliminary Procedures
<ol>
	<li>Glass capillary for solution injection. Using a capillary puller (Narishige, Tokyo, Japan), prepare a sharp pipette with a fine opening from a glass capillary micro tube (Brand GMBH, Wertheim, Germany). The size of the opening should be verified by passing air through the capillary to obtain fine bubbling in clean water. If the capillary is closed, then one may open it by breaking its edge. This may be accomplished by pressing or scratching it gently against the water containing tube walls. Prepare the capillary such that the opening is nearly blocked but is sufficiently open to allow the formation of sub-millimeter bubbles.</li>
	<li>Copper disc cleaning. Sonicate the copper discs for 10 min in 0.1% Micro-90 soap (Cole-Parmer, Vernon Hills, Illinois, USA), then wash with double distilled water. Introduce the discs into an isopropanol (technical) solution and sonicate again for 10 min. Finally, dry the discs using filtered air. This cleaning stage is critical to avoid IBP contamination between experiments.</li>
	<li>Double-layer coverglass assembly. A coverglass assembly was prepared to allow for sample observation without condensing moisture on the cover glass surface. This was achieved by placing a Drierite (W.A. Hammond Drierite, Xenia, Ohio, USA) particle (2 mm in diameter) between two coverslips that were then glued with a hot glue gun. This configuration prevented condensation that could block the view when the sample was cooled to low temperatures and removed the need to blow dry air onto the observation window.</li>
</ol>
1. Cooling Stage Set-up
<ol>
	<li>Connect the water flow inlet and outlet of the cooling stage to 4 mm inner diameter Tygon tubes (Saint-Gobain, Paris, France), and connect the water flow inlet tube to a water pump.</li>
	<li>Connect a 4 mm inner diameter Tygon tube to the inlet of the cooling stage to deliver dry air. The air was dried using an in-line Drierite column.</li>
	<li>Operate the air and water pumps. Note that the cooling elements should not be run without a heat sink.</li>
	<li>Turn on the temperature controller, camera, and LabVIEW routine.</li>
</ol>
2. Sample Preparation
<ol>
	<li>Place a 3-4 &mu;l droplet of immersion oil B (Cargille laboratories, Cedar Grove, New Jersey, USA) on the back side of a 7 mm diameter copper disc having 500 &mu;m holes drilled through the disc.</li>
	<li>Position the copper disc on the cooling stage with the immersion oil side facing down.</li>
	<li>Connect the capillary tube (the blunt edge) to a 0.7 mm inner diameter Tygon tube connected at the other end to a 2 ml glass syringe (Poulten-Graf, Wertheim, Germany).</li>
	<li>Prior to using the capillary tube, check the small opening of the capillary to ensure that the opening is an appropriate size (see the Preliminary procedures).</li>
	<li>Slowly insert the glass capillary into the prepared IBP protein sample tube (2.4 &mu;M MpIBP-GFP in 20 mM CaCl2 and 25 mM Tris-HCl at pH 8, see reference10 for the preparation details) and pull the glass syringe until the glass capillary contains 0.1 &mu;l of the protein solution.</li>
	<li>Begin video recording via the LabVIEW software.</li>
	<li>Insert the sharp edge of the glass capillary (containing the protein solution) into one of the holes in the copper disc on the cooling stage.</li>
	<li>While observing through the microscope (Olympus, Tokyo, Japan, 10x objective), carefully penetrate the immersion oil layer with the glass capillary tip, and press the glass syringe (very delicately) to deliver a small amount (~10 nl) of the protein solution to create a 200 &mu;m droplet.</li>
	<li>Cover the hole in the cooling stage with the double layer coverglass assembly (see the Preliminary Procedures).</li>
</ol>
3. TH Activity Measurement
<ol>
	<li>Press the cooling button and set the temperature to -40 &deg;C.</li>
	<li>Initially, the solution droplet will be clear. At low temperatures, typically in the range -30 &deg;C to -35 &deg;C, the droplet changes color, indicating that the solution has been frozen. Immediately after the sample has frozen, increase the temperature slowly until the bulk ice begins to melt. A gradual increase of the temperature is necessary to avoid overshooting of the temperature that could result in the complete melting of the sample.</li>
	<li>Switch to a 50x objective and begin to melt the ice by adjusting the temperature. This adjustment is interactive, and the final steps are typically performed using small temperature steps of 0.002 &deg;C. Continue to melt until a single crystal remains. The final size of the crystal should be around 10 &mu;m. The highest temperature at which melting has ceased is determined to be the melting point and is determined accurately in the later video analysis stage.</li>
	<li>Set the temperature to a few hundredths of a Celsius degree below the melting point of the crystal and begin a temperature ramp with a 10 min delay. Adjust the ramping rate as desired. During this time, the crystal will be exposed to the IBPs.</li>
	<li>Upon completion of the 10 min exposure time, the temperature will decrease automatically under the control of the LabVIEW routine.</li>
	<li>Observe the crystal shape as the temperature decreases. At some point, the sudden burst of the ice crystal may be observed. The temperature at which this occurs is noted as the crystal burst temperature.</li>
	<li>Use video analysis to determine the accurate melting point and the burst temperature. First, by using video analysis, find the accurate melting point. Recall that the highest temperature at which melting has ceased is determined to be the melting point. Document this melting point in a spreadsheet program. Then, determine the accurate crystal burst temperature, and document this value as well. The difference between the melting point and the freezing point, or crystal burst temperature, is the thermal hysteresis activity of the IBP solution.</li>
</ol>
4. Measurement of the Time-dependent TH Activity
<ol>
	<li>Follow the protocol described in Sections 3.1-3.3 to prepare a single crystal of ice.</li>
	<li>After formation of the crystal, set the delay time of the ramp as desired, and turn on the ramp.</li>
	<li>The temperature will decrease at a fixed rate (according to the operators' requirements) automatically once the ramp delay time has passed.</li>
	<li>Document the temperature at which the crystal burst occurs. Calculate the exposure time (the time between crystal formation and the crystal burst).</li>
	<li>Repeat the experiment for various delay times and plot the TH activity as a function of the exposure time to evaluate the time-dependence of the TH activity.</li>
</ol>

<ol>
	<li>DeVries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoproteins+as+biological+antifreeze+agents+in+antarctic+fishes.">Glycoproteins as biological antifreeze agents in antarctic fishes.</a> Science. 172, 1152-1155 (1971).</li><li>Worrall, D., Elias, L., Ashford, D., Smallwood, M., Sidebottom, C., Lillford, P., Telford, J., Holt, C., Bowles, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+carrot+leucine-rich-repeat+protein+that+inhibits+ice+recrystallization.">A carrot leucine-rich-repeat protein that inhibits ice recrystallization.</a> Science. 282, 115-117 (1998).</li><li>Raymond, J. A., Knight, C. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+adaptation+in+Arctic+and+Antarctic+fungi.">Cold adaptation in Arctic and Antarctic fungi.</a> New Phytol. 151, 341-353 (2001).</li><li>Gilbert, J. A., Hill, P. J., Dodd, C. E., Laybourn-Parry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+antifreeze+protein+activity+in+Antarctic+lake+bacteria.">Demonstration of antifreeze protein activity in Antarctic lake bacteria.</a> Microbiology. 150, 171-180 (2004).</li><li>Raymond, J. A., DeVries, A. 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L., Braslavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superheating+of+ice+crystals+in+antifreeze+protein+solutions.">Superheating of ice crystals in antifreeze protein solutions.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 5423-5428 (2010).</li><li>Gilbard, J. P., Farris, R. L., Santamaria, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmolarity+of+tear+microvolumes+in+keratoconjunctivitis+sicca.">Osmolarity of tear microvolumes in keratoconjunctivitis sicca.</a> Arch. Ophthalmol. 96, 677-681 (1978).</li><li>Gilbert, J. A., Davies, P. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+facile+method+for+determining+ice+recrystallization+inhibition+by+antifreeze+proteins.">A facile method for determining ice recrystallization inhibition by antifreeze proteins.</a> Biochem. Bioph. Res. Co. 311, 1041-1046 (2003).</li><li>Knight, C. A., Hallett, J., Devries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solute+Effects+on+Ice+Recrystallization+-+an+Assessment+Technique.">Solute Effects on Ice Recrystallization - an Assessment Technique.</a> Cryobiology. 25, 55-60 (1988).</li><li>Celik, Y., Drori, R., Pertaya-Braun, N., Altan, A., Barton, T., Bar-Dolev, M., Groisman, A., Davies, P. L., Braslavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+experiments+reveal+that+antifreeze+proteins+bound+to+ice+crystals+suffice+to+prevent+their+growth.">Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 1309-1314 (2013).</li><li>Middleton, A. J., Marshall, C. B., Faucher, F., Bar-Dolev, M., Braslavsky, I., Campbell, R. L., Walker, V. K., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifreeze+protein+from+freeze-tolerant+grass+has+a+beta-roll+fold+with+an+irregularly+structured+ice-binding+site.">Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site.</a> J. Mol. Biol. 416, 713-724 (2012).</li></ol>

No conflicts of interest declared.

This work demonstrates the operation of a computer-controlled nanoliter osmometer that enables accurate measurements of TH activity with extraordinary temperature control. In any temperature-sensitive system, unwanted temperature gradients must be avoided. To avoid temperature gradients in the apparatus presented here, the test solution droplet must be positioned in the center of a hole in the copper disc cooling stage (step 2.7). Additionally, the single crystal should be in the center of the droplet rather than near the edges (in most cases, this will happen spontaneously). The time dependence described indicates that the cooling rate may influence the TH readings. Thus, we suggest including a report of the time during which the crystal was exposed to the solution prior to cooling, as well as the cooling rate. We typically waited 10 min prior to ramping down the temperature at 0.01 &deg;C steps each 4 sec.
The LabVIEW-controlled cooling stage was adapted for use with an inverted microscope on which microfluidic devices could be thermally manipulated. This system facilitates the performance of solution exchange experiments involving ice crystals and IBPs tagged with eGFP9, 10, 16. The LabVIEW-controlled system may be adapted to a Clifton stage by connecting the 3,040 temperature controller via a designated adapting electric circuit. Such a system is operated in the Davies lab17. The LabVIEW software and the designated adapting electric circuit design for the Clifton stage are available upon request.
In conclusion, we describe a nanoliter osmometer that facilitates the sensitive control and manipulation of temperature and the rate of temperature increase and decrease (with 0.002 &deg;C sensitivity), coordinated with a video interface through a LabVIEW routine for real-time analysis. This system can perform reproducible rate-controlled experiments that are important for investigating the kinetics of IBP interactions with ice. Such experiments can address several long-debated issues surrounding the mechanism of action of IBPs.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number/model</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Immersion oil Type B</td>
 <td>Cargille Laboratories</td>
 <td>16484</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Drierite</td>
 <td>W.A. Hammond Drierite</td>
 <td>043063 2270g</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro 90 cleaning solution</td>
 <td>Cole-Parmer</td>
 <td><a href="http://www.coleparmer.com/Product/Cole_Parmer_small_sup_reg_sup_small_Micro_90_small_sup_reg_sup_small_cleaning_solution_case_of_four_4_Liter_bottles/EW-18100-11" target="_blank">EW-18100-11</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Capillary puller</td>
 <td>Narishige</td>
 <td>PB-7</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass capillary tubes</td>
 <td>Brand GNBH</td>
 <td>7493 21</td>
 <td>75 mm long, 1.15 diameter</td>
 </tr>
 <tr>
 <td>Temperature controller</td>
 <td>Newport, Irvine, California, United States</td>
 <td>Model 3040</td>
 <td>Model 3040</td>
 </tr>
 <tr>
 <td>Light microscope</td>
 <td>Olympus</td>
 <td>Model BH2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10x objective</td>
 <td>Olympus</td>
 <td>&nbsp;</td>
 <td>S Plan 10, 0.3, 160/0.17</td>
 </tr>
 <tr>
 <td>50x objective</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>CF plan, 50X/0.55 EPI ELWD</td>
 </tr>
 <tr>
 <td>CCD Camera</td>
 <td>Provideo</td>
 <td>CVC-140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tygon tubes</td>
 <td>Saint-Gobain, Paris, France</td>
 <td>&nbsp;</td>
 <td>Tygon Formulation S-50-HL Tubing</td>
 </tr>
 <tr>
 <td>Glass syringe (2 ml)</td>
 <td>Poulten-Graf, Wertheim, Germany</td>
 <td>7 10227</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GPIB-PCI card</td>
 <td>National instruments, Austin, Texas, USA</td>
 <td>778032-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Video frame grabber IMAQ-PCI-1407</td>
 <td>National instruments, Austin, Texas, USA</td>
 <td>322156B-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LabVIEW System Design Software</td>
 <td>National instruments, Austin, Texas, USA</td>
 <td>Version 8</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DiVx Author software</td>
 <td>DiVx LLC, San Diego, CA, USA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

labview-operated novel nanoliter osmometer for ice binding protein investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Measurement of the TH time dependence
The LabVIEW-operated nanoliter osmometer facilitates the performance of accurate TH activity measurements. The constant temperature reduction rate permitted the measurement of the TH time dependence. The precise temperature control enabled by the nanoliter osmometer was crucial for these experiments. The exposure time of an ice crystal to the IBPs in solution is defined as the time period from the formation of the crystal (the end of the melting process) until the sudden growth of ice around the crystal (crystal burst). We found that the exposure time of the ice crystals to the IBPs crucially affected the TH activity. Short periods of IBP exposure (a few seconds) produced a low TH activity in the MpIBP-GFP solution (2.4 &#956;M) (Figure 5). The TH activity increased with IBP exposure time until it reached a plateau at 4 min IBP exposure. At higher IBP concentrations, the plateau was reached at shorter times.
<img alt="Figure 1" src="/files/ftp_upload/4189/4189fig1.jpg" /> 
Figure 1. Schematic diagram illustrating IBPs adsorbed to ice. Adopted with permission from 10.
<img alt="Figure 2" fo:src="/files/ftp_upload/4189/4189fig2highres.jpg" src="/files/ftp_upload/4189/4189fig2.jpg" /> 
Figure 2. The cooling stage. A) Connected to tubes on the microscope. B) Without the upper lead. C) Schematic diagram. <a href="https://www.jove.com/files/ftp_upload/4189/4189fig2large.jpg" target="_blank">Click here to view larger figure </a>
<img alt="Figure 3" src="/files/ftp_upload/4189/4189fig3.jpg" /> 
Figure 3. Screenshot of the LabVIEW interface. <a href="https://www.jove.com/files/ftp_upload/4189/4189fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 4" fo:src="/files/ftp_upload/4189/4189fig4highres.jpg" src="/files/ftp_upload/4189/4189fig4.jpg" /> 
Figure 4. Temperature stability graph. The temperature controller was set to lower the temperature 0.01 &#176;C every 15 sec.
<img alt="Figure 5" fo:src="/files/ftp_upload/4189/4189fig5highres.jpg" src="/files/ftp_upload/4189/4189fig5.jpg" /> 
Figure 5. MpIBP TH activity as a function of ice crystal exposure time to the IBPs. Each time point is the average of 3-6 experiments.

Watch this Scientific Journal Video about LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations at JoVE.com

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

labview-operated-novel-nanoliter-osmometer-for-ice-binding-protein

Research

JoVE Journal

Chemistry

Surface Passivation for Single-molecule Protein Studies

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

We describe a method for passivating a glass surface using polyethylene glycol (PEG). This protocol covers surface cleaning, surface functionalization, and PEG coating. We introduce a new strategy for treating the surface with PEG molecules over two rounds, which yields superior quality of passivation compared to existing methods.

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important ...

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

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

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

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

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

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

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

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

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

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

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...