This work was supported by the Sogang University Research Grant of 201410036.

1. DNA Biotinylation
<ol>
	<li>Biotinylation of blunt ended DNA using Terminal Transferase (TdT) 
	Note: Use T4 DNA (166 kbp), which is a blunt ended DNA.
	<ol>
		<li>Add 5 &#181;l of 2.5 mM CoCl2, 5 &#181;l of 10&#215; reaction buffer, 0.5 &#181;l of 10 mM biotin-11-dUTP, 0.5 &#181;l of terminal transferase (10 units), and 0.5 &#181;l of T4 DNA (0.5 &#181;g/&#181;l) to the reaction mixture. Make to a final volume of 50 &#181;l by adding 38.5 &#181;l of water.</li>
		<li>Incubate the reaction mixture at 37 &#176;C for 1 hr. 
		Note: Extended reaction time may yield double-tethered DNA, i.e., it is possible that biotins are tagged at both ends.</li>
		<li>Stop the reaction by adding 5 &#181;l of 0.5 M EDTA, pH 8.</li>
		<li>Keep the tube at 4&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Biotinylation of sticky ended DNA Using DNA Ligase 
	Note: Use &#955; DNA (48.5 kbp), which is a sticky-end DNA.
	<ol>
		<li>Add 1 &#181;l of T4 DNA ligase, 5 &#181;l of 10&#215; ligation buffer, 1 &#181;l of 25 ng/&#181;l &#955; phage DNA, and add 43 &#181;l of water to make a final volume of 50 &#181;l.</li>
		<li>Keep the tube at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Functionalized Surface Derivatization
Note: In order to tether an end of a DNA molecule on glass surface, a primary amine group is silanized on a coverslip followed by biotin-PEG coating as shown in Figure 1. This PEGylation process is important for single-molecule DNA imaging because it can significantly decrease random noise created by attachment of undesired molecules on the surface.
<ol>
	<li>Piranha Cleaning 
	Note: Piranha solutions react vigorously with organic materials, therefore it should be handled with care, following proper safety guidelines.
	<ol>
		<li>Place coverslips on a polytetrafluoroethylene (PTFE) rack, and hold them by PTFE thread seal tape lengthwise, half on the edge of the surfaces and half on the rack. After wrapping, leave a long piece (~ 5 cm) of tape for handling the rack during the cleaning procedure.</li>
		<li>Fill a 1 L glass beaker with 350 ml of H2SO4 and 150 ml of H2O2 to make piranha solution in a fume hood. Place the rack in piranha solution for 2 hr.</li>
		<li>Empty piranha solution from the beaker, and rinse coverslips thoroughly with deionized water until the pH of water in the beaker reaches neutral. Use pH paper strips.</li>
		<li>Sonicate the racks of cover slips in the beaker containing water, for 30 min. Empty water from the beaker just before amino silanization. Use 75 W of sonication power to clean and derivatize the glass substrates.</li>
	</ol>
	</li>
	<li>Aminosilanization on Glass Surface
	<ol>
		<li>Add 2 ml of N-[3-(trimethoxysilyl)propyl]ethylenediamine and 10 ml of glacial acetic acid to 200 ml of methyl alcohol in a clean polypropylene container for preparing the aminosilanization solution.</li>
		<li>Place cleaned coverslips in the polypropylene container. Shake them at 100 rpm and room temperature for 30 min.</li>
		<li>Sonicate the beaker with derivatized cover slips for 15 min at 75 W. Shake them at 100 rpm and room temperature for at least 30 min.</li>
		<li>Empty the solution from the beaker, and rinse coverslips carefully, once with methyl alcohol, and twice with ethyl alcohol. Store coverslips in ethyl alcohol and use them within two weeks.</li>
	</ol>
	</li>
	<li>PEGylation of the coverslip
	<ol>
		<li>Make 10 ml of 0.1 M sodium bicarbonate (NaHCO3). Filter the solution with a 0.22-&#181;m syringe filter.</li>
		<li>Dissolve 2 mg of biotin-PEG-succinimidyl carbonate (biotin-PEG-SC) and 80 mg of PEG-succinimidyl valerate (mPEG-SVG) in 350 &#181;l of NaHCO3. Use a light protection tube.</li>
		<li>Vortex the tube vigorously for 10 sec, and centrifuge it at 10,000 &#215; g for 1 min to remove bubbles.</li>
		<li>Rinse a glass slide with acetone, followed by rinsing with ethyl alcohol. Allow the slide to air dry completely.</li>
		<li>Place a drop (50 &#181;l) of PEG solution on the clean glass slide. Cover the droplet gently with an amino silanized cover slip, without generating any bubbles.</li>
		<li>Place slides for 3 hr to overnight in a dark, well-leveled humid chamber at room temperature. Use an empty pipette tip holder as the chamber, with water at the bottom. Seal residual PEG solution in the tube and keep it at 4 &#176;C.</li>
		<li>Rinse the PEGylated coverslip thoroughly with deionized water. Store it in a dark and dry place until use.</li>
	</ol>
	</li>
</ol>
3. Assembling a Flow Chamber
<ol>
	<li>Fabricate a punched acrylic holder, as in Figure 2. Ensure that the diameter of the tubing insert is 0.762 mm, and designate one hole is an inlet and the other is an outlet (Figure 2).</li>
	<li>Place double-sided sticky tape strips (width 5-6 mm) on an acrylic holder (Figure 2), aligning perpendicularly to an inlet and outlet hole, not perturbing any channel holes. Use these tape strips as multi-channel walls to make chambers. Scrub the tape using a pipet tip.</li>
	<li>Place a PEGylated coverslip on the top to make flow chambers, with the PEGylated side facedown.</li>
	<li>To prevent any solution from leaking, press the top of a coverslip over the area where double-sided tapes are placed. Add quick-dry epoxy glue to close the edges of the chamber at the top and at the bottom (yellow color in Figure 2).</li>
	<li>Connect a short piece (2.5 cm) of tubing (outer diameter, OD, 0.042&#34;) to a gas-tight syringe and seal the joint with epoxy glue.</li>
	<li>Connect a long flexible tubing (OD: 0.03&#34;) to the tubing linked to the syringe and seal the joint with epoxy glue.</li>
	<li>Fill the tubing linked to the syringe with DI water. Make sure there is no air bubble.</li>
	<li>Insert the tubing into the hole of the flow chamber sealed with epoxy glue.</li>
	<li>Place a yellow tip (200 &#181;l tip) on the other hole as a reservoir.</li>
</ol>
4. Sample Loading into Flow Chamber
Note: Neutravidin can be replaced with other avidin proteins such as streptavidin. All of the reactions can be performed at room temperature, unless it is mentioned. Take sample solution in a yellow tip, and install the tip with the solution onto the hole of acrylic holder (Figure 2b).
<ol>
	<li>Set the flow rate of the syringe pump at 50 &#181;l/min. Load 20 &#181;l of avidin protein (25 &#181;g/ml in T50 solution, Tris 10 mM, NaCl 50 mM, pH 8), and keep it for 10 min.</li>
	<li>Load 20 &#181;l of biotinylated oligodeoxynucleotides (100 &#181;M in 1&#215; TE), and keep it for 10 min. If terminal transferase is used, skip the loading of oligodeoxynucleotides.</li>
	<li>Load 20 &#181;l of DNA solution from Step 1 into the flow chamber at a flow rate of 10 &#181;l/min, and keep it for 30 min.</li>
	<li>Wash the flow chamber with 1&#215; TE, and load 40 &#181;l of FP-DBP10 (~80 nM).</li>
	<li>Observe DNA under a fluorescent microscope with continuous flow of staining molecules in 1&#215; TE on a 60X objective lens. Use 488 nm solid state laser for excitation of FP(eGFP)-DBP. For full stretch of DNA molecules, apply different flow rates according to the DNA lengths used. For example, apply 50 &#181;l/min for 48.5 kbp of &#955; DNA, and 100 &#181;l/min for 166 kbp of T4 DNA.
	<ol>
		<li>For recycling of the acrylic holder, soak assembled flow chambers in a household detergent solution12. Remove tapes and epoxy with a razor blade and rub with hands to completely take them off. Unclog holes with syringe needles. Keep the holders in deionized water until further use.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Smith, S. B., Finzi, L., Bustamante, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Mechanical+Measurements+of+the+Elasticity+of+Single+DNA-Molecules+by+Using+Magnetic+Beads.">Direct Mechanical Measurements of the Elasticity of Single DNA-Molecules by Using Magnetic Beads.</a> Science. 258 (5085), 1122-1126 (1992).</li><li>Finkelstein, I. J., Visnapuu, M. -. L., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+imaging+reveals+mechanisms+of+protein+disruption+by+a+DNA+translocase.">Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase.</a> Nature. 468 (7326), 983-987 (2010).</li><li>Doyle, P. S., Ladoux, B., Viovy, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+a+tethered+polymer+in+shear+flow.">Dynamics of a tethered polymer in shear flow.</a> Phys. Rev. Lett. 84 (20), 4769-4772 (2000).</li><li>Perkins, T. T., Quake, S. R., Smith, D. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relaxation+of+a+Single+DNA+Molecule+Observed+by+Optical+Microscopy.">Relaxation of a Single DNA Molecule Observed by Optical Microscopy.</a> Science. 264 (5160), 822-826 (1994).</li><li>Cai, W., 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=Ordered+restriction+endonuclease+maps+of+yeast+artificial+chromosomes+created+by+optical+mapping+on+surfaces.">Ordered restriction endonuclease maps of yeast artificial chromosomes created by optical mapping on surfaces.</a> Proc. Natl. Acad. Sci. U.S.A. 92 (11), 5164-5168 (1995).</li><li>Kim, Y., Jo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutravidin+coated+surfaces+for+single+DNA+molecule+analysis.">Neutravidin coated surfaces for single DNA molecule analysis.</a> Chem. Comm. 47 (22), 6248-6250 (2011).</li><li>Kim, Y., 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=Nanochannel+confinement:+DNA+stretch+approaching+full+contour+length.">Nanochannel confinement: DNA stretch approaching full contour length.</a> Lab on a Chip. 11 (10), 1721-1729 (2011).</li><li>Glazer, A. N., Rye, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+dye-DNA+intercalation+complexes+as+reagents+for+high-sensitivity+fluorescence+detection.">Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence detection.</a> Nature. 359 (6398), 859-861 (1992).</li><li>Graneli, A., Yeykal, C. C., Prasad, T. K., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organized+arrays+of+individual+DNA+molecules+tethered+to+supported+lipid+bilayers.">Organized arrays of individual DNA molecules tethered to supported lipid bilayers.</a> Langmuir. 22 (1), 292-299 (2006).</li><li>Lee, S., 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=DNA+binding+fluorescent+proteins+for+the+direct+visualization+of+large+DNA+molecules.">DNA binding fluorescent proteins for the direct visualization of large DNA molecules.</a> Nucleic Acids Res. , (2015).</li><li>Roy, R., Hohng, S., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+single-molecule+FRET.">A practical guide to single-molecule FRET.</a> Nat Methods. 5 (6), 507-516 (2008).</li><li>Chandradoss, S. D., 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=Surface+passivation+for+single-molecule+protein+studies.">Surface passivation for single-molecule protein studies.</a> J. Vis. Exp. (86), e50549 (2014).</li></ol>

The authors have nothing to disclose.

Here we present a platform for visualizing long DNA molecules biotinylated for anchoring on surfaces. We have reported an approach for DNA molecules tethered on an avidin protein coated surface with biotinylated bovine serum albumin.6 In the earlier approach, we found a crucial issue of DNA photo-cleavage caused by bis-intercalation dyes that stain DNA molecules tethered on the surface. As these continually excited fluorophores have a high probability to attack a DNA phosphate backbone,9 the excitation light power had to be minimized to avoid photo-cleavage.
In order to overcome this inconvenience, we developed genetically engineered fluorescent proteins with DNA-binding capability (FP-DBP) for staining DNA without photo-cleavage.10 We observed reliable DNA staining on avidin-coated protein surfaces. Nonetheless, this platform had another issue of undesirable aggregation of fluorescent proteins on the protein surface, resulting in increased background noise. It is essential to minimize this random noise for single-molecule DNA analysis to enhance the contrast of DNA and proteins from the background. The use of FP-DBP therefore requires surface passivation to prevent the fluorescent protein from being adsorbed on the surface. PEG could be a powerful candidate for this purpose.12 Hence, we used biotinylated-PEG, which dramatically enhanced signal-to-noise ratio for imaging FP-DBP stained DNA molecules tethered on the surface.
There are several critical steps and notes in this method for higher yields. In order to tether an end of a DNA molecule on a glass surface, a primary amine group is silanized on a coverslip followed by biotin-PEG coating as shown in Figure 1. This surface passivation process is critical for single-molecule DNA imaging because it can significantly decrease noise-generating random adsorption on the surfaces. Therefore, overnight aminosilanization and PEGylation are strongly recommended. In addition, cleaning the glass slide as well as the cover glass with piranha solution can further reduce background noise.
A biotinylated oligonucleotide must have a phosphate group at 5&#39; end in order to link it to 3&#39; hydroxyl group of a large DNA molecule. The sequence of biotinylated oligonucleotide is 5&#39;-p-GGGCGGCGACCT-TEG-Biotin-3&#39; for &#955; DNA tethering. The &#955; DNA solution has to be loaded shortly after the preparation of &#955; DNA solution (described in 1.1), because &#955; DNA often forms concatemers with long-time storage. For blunt-ended DNA such as T4 DNA, terminal transferase can add biotinylated dUTPs at both ends of DNA without any specificity. Thus, terminal transferase reaction time has to be carefully adjusted around an hour, otherwise extended reaction may yield double-labeled DNA (i.e., both ends are labeled with biotin). In addition to viral DNA such as &#955; and T4, any types of DNA can be utilized in this platform. However, it is recommended to use fragments from gigantic genomic DNA after digestion by rare-cutter restriction enzymes such as SwaI or NotI.
Different flow rates are applied for different lengths of DNA. Usually, longer DNA molecules require faster flow rates to fully stretch the DNA molecules, to match the exact contour length. For example, &#955; DNA can stretch up to 16.5 &#181;m, which corresponds to the calculated value of 0.34 nm x 48,502 bp.
It is quite important to freshly prepare all materials, particularly all of proteins and PEGs. For example, Neutravidin readily loses its function of binding biotins if it is stored as a diluted solution. For PEG solutions, pH is critical to conjugate functional groups of a primary amine and n-hydroxysuccinimide. Therefore, it is recommended to freshly prepare all materials at least every week.
Visualization of large DNA molecules tethered on the surface is a versatile platform to be applied for a variety of applications such as genomics, epigenomics, biochemical studies for DNA and protein interaction, and polymer physics studies. However, this approach is currently limited only to model systems using relatively simple and well-known DNA molecules such as the viral genome. To be extended to the human genome and epigenome, it is critically important to establish a robust, reliable, and simple platform for practical use. By overcoming light-induced DNA photo-cleavage and preventing random adsorption of fluorescent protein on the surface, this platform therefore provides clear and high-contrast images for DNA elongated with shear flows.

Visualization of large DNA molecules tethered on glass or bead surfaces has been utilized for investigating DNA-protein interactions, protein dynamics on DNA substrate,1,2 and polymer physics.3,4 A platform for single-tethered large DNA molecules has a few distinct advantages compared to other DNA immobilization methods.5 First, a large DNA molecule tethered on the surface has a natural random-coil conformation without a shear flow, which is critically important for a DNA-binding protein to recognize its binding site. Second, it is very easy to change the chemical environment around DNA molecules for a series of enzymatic reactions in a flow chamber. Third, a microfluidic shear flow induces DNA molecular stretching up to 100% of the full contour length, which is very difficult to achieve using alternative DNA elongation approaches such as surface immobilization6 and nanochannel confinement.7 A fully stretched DNA molecule also provides positional information that can be useful for monitoring enzymatic movements on the genomic map.
Nevertheless, the DNA tethering approach has a critical shortcoming in that the intercalating dye such as YOYO-1 generally causes tethered DNA molecules to be readily broken by fluorescence excitation light. Generally, large DNA molecules have to be stained with a fluorescent dye for visualization under a fluorescent microscope. For this purpose, YOYO-1 or other TOTO series dyes are primarily used because these dyes fluoresce only when they intercalate double-stranded DNA.8 However, it is well-known that bis-intercalating dye causes light-induced DNA photo-cleavage because of the intercalation of fluorophores.9 Further, fluorescently stained DNA molecules tethered on the surface are more fragile since shear flows can exert breaking forces on freely moving DNA molecules. Therefore, we developed FP-DBP as a novel DNA-staining protein dye for imaging large DNA molecules tethered on the surface. The advantage of using FP-DBP is that it does not cause photo-cleavage of the DNA molecules to which it binds.10 In addition, FP-DBP does not increase the contour length of DNA, while bis-intercalating dyes increase the contour length by about 33%.
This video method introduces the experimental approach for tethering large DNA molecules to a PEG-biotin surface. Figure 1 illustrates different approaches of tethering DNA with blunt ends and sticky ends. Thus, this staining method can be applied to any type of DNA molecule. Figure 2 depicts a schematic representation of the flow chamber assembly that can be controlled by a syringe pump to generate shear flows to stretch DNA molecules as well as to load chemical and enzyme solutions. Figure 3 demonstrates micrographs of fully stretched DNA molecules tethered on the PEGylated surface11 and stained with FP-DBP.

<table border="0" cellpadding="0" cellspacing="0" width="925">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">1. DNA Biotinylation</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">1.1) Biotinylation of blunt-end DNA using Terminal Transferase (TdT)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Terminal Transferase</td>
			<td style="width:167px;">New England Biolabs</td>
			<td style="width:133px;">M0315S</td>
			<td>Provided with 10x reaction buffer, 2.5 mM Cobalt chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Biotin-11-dUTP</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:133px;">R0081</td>
			<td>Biotin-ddNTP is also available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">T4GT7 Phage DNA</td>
			<td style="width:167px;">Nippon Gene</td>
			<td style="width:133px;">318-03971</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:133px;">E6758</td>
			<td>EDTA</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">1.2) Biotinylation of sticky end DNA Using DNA Ligase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">T4 DNA Ligase</td>
			<td style="width:167px;">New England Biolabs</td>
			<td style="width:133px;">M0202S</td>
			<td>Provided with 10x reaction buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">Lambda Phage DNA</td>
			<td style="width:167px;">Bioneer</td>
			<td style="width:133px;">D-2510</td>
			<td>Also available at New England Biolabs</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:925px;">2. Functionalized Surface Derivatization</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">2.1) Piranha Cleaning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Coverslip</td>
			<td style="width:167px;">Marienfeld-Superior</td>
			<td style="width:133px;">0101050</td>
			<td>22x22 mm, No. 1 Thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Teflon rack</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td>Custom Fabrication</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">PTFE Thread Seal Tape</td>
			<td style="width:167px;">Han Yang Chemical Co. Ltd.</td>
			<td style="width:133px;">3032292</td>
			<td>Teflon&#8482; tape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Sulferic acid</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">S280823</td>
			<td>H2SO4, 95 % Purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hydrogen peroxide</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">H290423</td>
			<td>H2O2, 35 % in water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Sonicator</td>
			<td style="width:167px;">Daihan Scientific Co. Ltd.</td>
			<td style="width:133px;">WUC-A02H</td>
			<td>Table-top Ultrasonic Cleaner</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">2.2) Aminosilanization on Glass Surface</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">N-[3-(Trimethoxysilyl)propyl] 
			ethylenediamine</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:133px;">104884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Glacial Acetic Acid</td>
			<td style="width:167px;">Duksan Chemicals</td>
			<td style="width:133px;">414</td>
			<td>99 % Purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Methyl Alcohol</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">M300318</td>
			<td>99.9 % Purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Polypropylene Container</td>
			<td style="width:167px;">Qorpak</td>
			<td style="width:133px;">PLC-04907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Ethyl Alcohol</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">A300202</td>
			<td>99.9 % Purity</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">2.3) PEGylation of the coverslip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Sodium Bicarbonate</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:133px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Syringe Filter</td>
			<td style="width:167px;">Sartorius</td>
			<td style="width:133px;">16534----------K</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Biotin-PEG-SC</td>
			<td style="width:167px;">Laysan Bio</td>
			<td style="width:133px;">Biotin-PEG-SC-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">mPEG-SVG</td>
			<td style="width:167px;">Laysan Bio</td>
			<td style="width:133px;">MPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acetone</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">A300129</td>
			<td>99 % Purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Microscope Slides</td>
			<td style="width:167px;">Marienfeld-Superior</td>
			<td style="width:133px;">1000612</td>
			<td>~76x26x1 mm</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">3. Assembling a Flow Chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acrylic Support</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td>Custom Fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Double-sided Tape</td>
			<td style="width:167px;">3M</td>
			<td style="width:133px;"></td>
			<td>Transparent type</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Quick-dry Epoxy</td>
			<td style="width:167px;">3M</td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Polyethylene Tubing</td>
			<td style="width:167px;">Cole-Parmer</td>
			<td style="width:133px;">06417-11, 06417-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Gas Tight 250 &#181;l Syringe</td>
			<td style="width:167px;">Hamilton</td>
			<td style="width:133px;">81165</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Syringe Pump</td>
			<td style="width:167px;">New Era Pump Systems Inc.</td>
			<td style="width:133px;">NE-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">4. Sample Loading into Flow Chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Neutravidin</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:133px;">31000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Tris base</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:133px;">T1503-5KG</td>
			<td>Trizma base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:133px;">IX70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">EMCCD Camera</td>
			<td style="width:167px;">Q Imaging</td>
			<td style="width:133px;">Rolera EM-C2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Solid-state Laser (488 nm)</td>
			<td style="width:167px;">Oxxius</td>
			<td style="width:133px;">LBX488</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Alconox</td>
			<td style="width:167px;">Alconox Inc.</td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of surface-tethered large dna molecules with a fluorescent protein dna binding peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

Figure 1 shows two different DNA tethering methods depending on terminal structures of the DNA molecule. Figure 1a illustrates how the sticky ended DNA molecules are hybridized with complementary biotinylated oligonucleotides, which are immobilized on the avidin-coated PEG surface. Figure 1b shows the addition of biotinylated ddNTP or dNTP to the 3&#39; hydroxyl group of a blunt ended DNA by terminal transferase. We added flexible linkers between DNA molecules and biotin. This increased the ligation and avidin-biotin binding efficiency to provide sufficient space for the reactions. Figure 1c demonstrates the overall schematic representation for DNA tethering on the avidin-coated PEG surface.
Figure 2 depicts the assembly of the flow chamber along with an acrylic holder. Compared to glass/quartz slide glasses, a custom-made acrylic holder simplifies the preparation of the flow chamber for epi-fluorescent microscopy.12 The flow chamber enables instant changing of buffer conditions for enzyme reactions,6 and stretching DNA molecules.
Figure 3 demonstrates single-tethered DNA and T4 &#955;DNA stained with FP-DBP. Microfluidic shear flows elongated single DNA molecules up to full contour lengths such as 16.5 &#956;m (48.5 kb &#955; DNA x 0.34 nm) and 56.4 &#956;m (166 kb T4 DNA x 0.34 nm). We were able to repeat DNA stretching and relaxation multiple times during an extended period (e.g., half-hour), since we utilized FP-DBP that does not cause photo-cleavage.
<img alt="Figure 1" src="/files/ftp_upload/54141/54141fig1.jpg" /> 
Figure 1. Schematic illustration of DNA tethering. a) A sticky ended DNA is hybridized with a biotinylated complementary oligonucleotide. Triethylene glycol is a flexible linker between the oligonucleotide and biotin. b) Biotinylated dNTP is added to a blunt end of double stranded DNA, by terminal transferase. Polycarbon spacer (11-atoms) is a flexible linker between dNTP and biotin. c) Biotinylated DNA is anchored to an avidin protein, which is linked to a biotinylated PEG covalently bonded to the glass surface. The ratio of biotinylated PEG to normal PEG was 0.025 (1:40). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54141/54141fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54141/54141fig2.jpg" /> 
Figure 2. Flow chamber. a) Flow chamber consisting of an acrylic acid resin holder, strips of double-sided tapes and a PEGylated cover slip. The acrylic holder of dimensions 76 mm x 26 mm x 5 mm (L x W x H), was fabricated with laser cutting. b) Side view of the Flow chamber: punched holes are an inlet port on which a yellow tip is installed for a buffer reservoir, and an outlet port that is connected to a tubing controlled by a syringe-pump. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54141/54141fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54141/54141fig3.jpg" /> 
Figure 3. Fluorescent microscopic images of stretched DNA molecules stained with FP-DBP. a) Bacteriophage &#955; DNA (48.5 kbp) tethered on the surface after ligation with biotinylated complementary oligonucleotides. b) Bacteriophage T4 DNA (166 kbp) tethered on the surface after adding biotin with terminal transferase. Indicated arrows show DNA molecules stretched up to their full contour lengths such as 16.5 &#181;m for &#955; DNA and 56.4 &#181;m for T4 DNA. Scale bars: 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54141/54141fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide at JoVE.com

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

visualization-surface-tethered-large-dna-molecules-with-fluorescent

Nanyang Technological University

Research

JoVE Journal

Biology

10.7K Views.  Sogang University. We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows. 

Video: Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...