This work was supported by Fondo Nacional de Desarrollo Cient&#237;fico y Tecnol&#243;gico (Fondecyt), Chile, Project 11130263 (to CW), Project CONICYT + NERC + Programa de Colaboraci&#243;n Internacional PCI-PII20150073 (to CW) and U-inicia from the Vicerrector&#237;a de Investigaci&#243;n Universidad de Chile (to CW). 
Thanks to Tatiana Naranjo-Palma for the help in the initial stage of this Project, Dr. Jorge Babul and Diego Quiroga-Roger for helpful discussion, Robert Lesch for revising the manuscript and Direcci&#243;n de extensi&#243;n de la Facultad de Ciencias Qu&#237;micas y Farmac&#233;uticas from Universidad de Chile. Thanks too to Marcelo P&#233;rez for editing the video and Neil Stevens for correcting the text and Errol Watson for providing the voice-over.

1. Preparing and Running the Gel
<ol>
	<li>Prepare the non-denaturing 12% polyacrylamide gel gels using the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) recipe found in Sambrook and Russell14 but using water instead of SDS.
	<ol>
		<li>To prepare 5 mL of resolving gel, use 1.7 mL of distilled water, 2 mL of acrylamide/bis acrylamide (30/0.8% w/v), 1.3 mL of 1.5 M Tris pH 8.8, 0.05 mL of 10% ammonium persulfate, and 0.002 mL of TEMED.</li>
		<li>To prepare 2 mL of stacking gel use 1.5 mL of water, 0.33 mL of acrylamide/bis acrylamide (30/0.8% w/v), 0.25 mL of 1 M Tris pH 6.8, 0.02 mL of 10% ammonium persulfate, and 0.002 mL of TEMED. The dimensions of the gel are 7.3 cm x 8.2 cm x 0.075 cm (l x w x d). 
		Note: Use a 15 well comb of 0.75 mm thickness for the best sensitivity.</li>
	</ol>
	</li>
	<li>Add the appropriate amounts of 6x loading buffer and DNA sample to a 1.5 mL microcentrifuge tube (e.g. use 1 &#181;L of 6x loading buffer to 5 &#181;L of DNA sample).</li>
	<li>Place the gel in the vertical electrophoresis chamber.
	<ol>
		<li>Fill the chamber with 750 mL of 1x TAE buffer to run 2 gels. To prepare 1 L of 50x TAE, use 442 g of Tris base, 57.1 mL of glacial acetic acid, and 100 mL of 0.5 M EDTA pH 8. Add distilled water to a final volume of 1 L.</li>
		<li>Load the samples in the gel. Run the gel at 100 V for 2 h.</li>
	</ol>
	</li>
	<li>Alternatively, prepare a 1x TAE agarose gel using the recipe found in Sambrook14.
	<ol>
		<li>Add 300 mL of 1x&#160;TAE to the horizontal electrophoresis chamber. Run the gel for 45 min at 100 V.</li>
	</ol>
	</li>
</ol>
2. NBT-SG I Staining (Figure 1).
<ol>
	<li>Acrylamide Gel Staining.
	<ol>
		<li>Prepare the solutions: 0.1% w/v NBT and 1.2x SG I. 
		Note: It is possible to use ethidium bromide, GelRed and SYBR Gold instead of SG I, but SG I had the best performance.</li>
		<li>Pour 16.75 mL of 1.2x SYBR Green I solution into a container of appropriate size. 
		Note: We used the lid of a pipette tip box as a container, and these volumes are calculated to cover the gel using this container; its dimensions are 11.6 cm x 7.6 cm x 2.6 cm (l x w x d).</li>
		<li>Place the gel in the solution. Add 3.25 mL of 0.1% w/v NBT solution to give 0.2 mM NBT and 1x (equivalent to 2.0 &#181;M) SG I final concentrations.</li>
		<li>Seal the container with plastic film, and completely wrap the container with aluminum foil to block any ambient light. 
		Note: The plastic film is used to prevent splashing and to avoid reaction of the aluminum foil with the staining solution.</li>
		<li>Shake for 25 min on an orbital shaker at 60 rpm.</li>
		<li>Remove aluminum and plastic cover. Expose to sunlight for 90 min maximum. Check every 5 min until the band of interest appears.</li>
		<li>To use blue light, prepare a protoboard with 3 Light-Emitting Diodes (LED) and place the LEDs about 5 mm from the gel surface (check the staining continuously, as the time needed will depend on the intensity of the LED light source).</li>
	</ol>
	</li>
	<li>Agarose Gel Staining.
	<ol>
		<li>After preparing and running a 1% agarose gel as described in sections 1.4 (the dimensions are 1 cm x 6.2 cm x 7 cm; l x w x d), follow the same steps as in 2.1 with these modifications:</li>
		<li>Pour 41.9 mL of 1.2x SG I solution into the lid of a pipette tip box; its dimensions are 11.6 cm x 7.6 cm x 2.6 cm (l x w x d). Add 8.1 mL of 0.1% NBT solution.</li>
		<li>Seal the container with plastic film, and then completely wrap the container with aluminum foil to block any ambient light.</li>
		<li>Shake for 1 h on an orbital shaker at 60 rpm.</li>
	</ol>
	</li>
</ol>
3. Data Analysis.
<ol>
	<li>NBT-SG I Calibration Curves.
	<ol>
		<li>Obtain gel images by scanning at 1200 dpi.</li>
		<li>Open software for image analysis. Straighten the images using an image editor. Run an image densitometry.</li>
		<li>Calculate the area under the curve. Prepare a graph of Signal/SignalMax versus DNA concentration.</li>
	</ol>
	</li>
</ol>
4. DNA Recovery.
<ol>
	<li>Prepare a 1% agarose gel in 1x TAE using the recipe found in Sambrook14.</li>
	<li>Add 300 mL of 1x&#160;TAE to the horizontal electrophoresis chamber. Load 4 &#181;L of 100 ng/&#181;L pDJ10015 plasmid and DNA ladder following the scheme given in Figure 2. Run the gel for 1 h at 100 V.</li>
	<li>Mark and weigh two 2 mL microcentrifuge tubes.</li>
	<li>Cut the gel down the middle in order to have two identical halves. Each one should contain the ladder and the sample plasmid to compare the different staining techniques. A schematic representation is in Figure 2.</li>
	<li>Stain one half with NBT-SG I as in step 2.
	<ol>
		<li>Cut out the plasmid band using a clean scalpel. Save the gel piece in one tube and weigh.</li>
	</ol>
	</li>
	<li>Stain the other part of the gel with SG I with 50 mL of 1x SG I solution and slowly shake (60 rpm) the gel for 1 h in darkness.
	<ol>
		<li>Place the gel on a transilluminator and expose for 2 min. Cut out the plasmid band using a clean scalpel.</li>
		<li>Save the gel piece in the other tube and weigh. 
		Note: The protocol can be paused here while keeping the tubes at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Carry out plasmid extraction using a commercial gel extraction kit.</li>
</ol>
5. Plasmid Normalization.
<ol>
	<li>In a 1.5 mL microcentrifuge tube, add 10 &#181;L of extracted plasmid and 60 &#181;L of type I water. Mix and transfer to a 60 &#181;L quartz cuvette or use 2 &#181;L directly in a Nanodrop.</li>
	<li>Measure absorption spectra between 230 to 320 nm. Calculate the sample&#39;s plasmid concentration using the following formula: 
	<img alt="Equation 1" src="/files/ftp_upload/56528/56528eq1v3.jpg" /> 
	where D is the dilution factor applied (in this case it is 70/10), Ax is absorption at x nm, and 50 is the conversion factor to &#181;g/mL for dsDNA.</li>
	<li>Dilute samples to 9.1 ng/&#181;L.</li>
	<li>Transform Escherichia coli (E. coli) competent cells (e.g. XL10 Gold) following the Inoue transformation procedure16 using 5 &#181;L of dilute sample.</li>
	<li>Count the number of colonies on the Petri dishes and note the dilution. Calculate the Colony Forming Units (CFU) with the following formula17: 
	<img alt="Equation 2" src="/files/ftp_upload/56528/56528eq2v2.jpg" /> 
	where D is the dilution factor applied from the initial culture.</li>
	<li>Perform an unpaired t-test.</li>
</ol>

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E., Yue, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+SYBR+Green+I+nucleic+acid+gel+stain+mutagenicity+and+ethidium+bromide+mutagenicity+in+the+Salmonella/mammalian+microsome+reverse+mutation+assay+(Ames+test).">Comparison of SYBR Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test).</a> Mutat Res. 439 (1), 37-47 (1999).</li><li>Xin, X., Yue, S., Wells, K. S., Singer, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SYBR+Green-I:+a+new+fluorescent+dye+optimized+for+detection+picogram+amounts+of+DNA+in+gels.">SYBR Green-I: a new fluorescent dye optimized for detection picogram amounts of DNA in gels.</a> Biophys. J. 66 (A150), (1994).</li><li>Flores, N., Valle, F., Bolivar, F., Merino, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+DNA+from+agarose+gels+stained+with+methylene+blue.">Recovery of DNA from agarose gels stained with methylene blue.</a> Biotechniques. 13 (2), 203-205 (1992).</li><li>Yang, Y. I., Jung, D. W., Bai, D. G., Yoo, G. S., Choi, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counterion-dye+staining+method+for+DNA+in+agarose+gels+using+crystal+violet+and+methyl+orange.">Counterion-dye staining method for DNA in agarose gels using crystal violet and methyl orange.</a> Electrophoresis. 22 (5), 855-859 (2001).</li><li>Yang, Y. I., 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=Detection+of+DNA+using+a+visible+dye,+Nile+blue,+in+electrophoresed+gels.">Detection of DNA using a visible dye, Nile blue, in electrophoresed gels.</a> Anal. Biochem. 280 (2), 322-324 (2000).</li><li>Santill&#225;n-Torres, 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=A+novel+stain+for+DNA+in+agarose+gels.">A novel stain for DNA in agarose gels.</a> Trends Genet. 9 (2), 40 (1993).</li><li>Adkins, S., Burmeister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+DNA+in+agarose+gels+as+migrating+colored+bands:+applications+for+preparative+gels+and+educational+demonstrations.">Visualization of DNA in agarose gels as migrating colored bands: applications for preparative gels and educational demonstrations.</a> Anal. Biochem. 240 (1), 17-23 (1996).</li><li>Cong, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+visible+dye-based+staining+method+for+DNA+in+polyacrylamide+gels+by+ethyl+violet.">A visible dye-based staining method for DNA in polyacrylamide gels by ethyl violet.</a> Anal. Biochem. 402 (1), 99-101 (2010).</li><li>Altman, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazolium+salts+and+formazans.">Tetrazolium salts and formazans.</a> Prog. Histochem. Cytochem. 9 (3), 1-56 (1976).</li><li>Nineham, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemistry+of+formazans+and+tetrazolium+salts.">The chemistry of formazans and tetrazolium salts.</a> Chem. Rev. 55 (2), 355-483 (1955).</li><li>Crandall, I. E., Zhao, B., Vlahakis, J. 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R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: a Laboratory Manual. , (2012).</li><li>Hansen, W., Garcia, P. 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The authors have nothing to disclose.

A sensitive and novel DNA staining method based on the reduction of NBT and the use of SG I was presented in this article. The critical step in this protocol is the incubation time of the gel in the NBT-SG I solution and the concentration of NBT. The staining time for agarose gels is longer than for acrylamide gels because of the greater thickness of agarose gels. This method does not work well in the presence of SDS as NBT precipitates in contact with it. If the gel obtains a purple background, we recommend that another...

With the advances in biochemistry and molecular biology, the studies involving DNA have required better techniques to analyze DNA. The first method for DNA staining was silver staining, which is very sensitive but lacks selectivity and does not allow sample recovery. Later, the development of fluorescent DNA staining allowed the selective quantification of DNA with the possibility of sample recovery. One of the first fluorescent dyes used for DNA quantification was ethidium bromide1, which is mutagenic2. However, now there are improved fluorescent dyes that are safer and more sensitive, such as GelRed and SYBR Green I (SG I)3; but all of these fluorescent dyes require the use of an ultraviolet (UV) transilluminator or fluorimeter.
There are other techniques for visibly staining DNA, such as methylene blue4 and crystal violet5,6,7,8,9, but all of these suffer from reduced sensitivity and selectivity.The tetrazolium salts are organic compounds susceptible to reduction, and when this happens they form an insoluble and colored formazan precipitate10,11. Recently, some bivalent tetrazolium salts have been shown to bind to DNA due to their positive tetrazolium rings12.
In a recent publication13, a new visible technique to stain and quantify DNA in polyacrylamide gels was proposed, using the reduction of a bivalent tetrazolium salt called nitro blue tetrazolium (NBT) and fluorescent dyes like ethidium bromide, GelRed, SYBR Green I and SYBR Gold. This reaction worked in the presence of blue light or sunlight and allowed sample recovery with improved quality compared to the use of SG I with a UV transilluminator.The objective of this paper is to provide a detailed protocol of the staining technique using tetrazolium salts.

<table border="0" cellpadding="0" cellspacing="0" width="785">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Nitro blue tetrazolium</td>
			<td style="width:227px;">Gold Biotechnology</td>
			<td style="width:307px;">298-83-9</td>
			<td style="width:64px;">reagent used in the staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">SYBR Green I 10000X</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">S7563</td>
			<td>reagent used in the staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Gel extraction kit</td>
			<td style="width:227px;">QIAGEN</td>
			<td style="width:307px;">28704</td>
			<td>used to extract plasmid from the gel in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">DNA ladder</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">SM 1331</td>
			<td>used to make calibration curves and as reference to cut band in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Agar Agar</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;"></td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">sodium chloride</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1064045000</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">yeast extract</td>
			<td style="width:227px;">Becton, Dickinson and Company</td>
			<td style="width:307px;">212750</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">peptone</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">72161000</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">TEMED</td>
			<td style="width:227px;">AMRESCO</td>
			<td style="width:307px;">761</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">ammonium persulfate</td>
			<td style="width:227px;">Calbiochem</td>
			<td style="width:307px;">2310</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">acrylamide</td>
			<td style="width:227px;">invitrogen</td>
			<td style="width:307px;">15512-023</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">bisacrylamide</td>
			<td style="width:227px;">SIGMA</td>
			<td style="width:307px;">146072</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Tris-base</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1083821000</td>
			<td>used to make TAE buffer and non-denaturing polyacrylamide gel tris-HCl buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">EDTA</td>
			<td style="width:227px;">J.T. Baker</td>
			<td style="width:307px;">8993-01</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Acetic acid</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1,000,632,500</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">agarose</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">16802</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">spectrophotometer</td>
			<td style="width:227px;">Tecan</td>
			<td style="width:307px;">infinite 200 pro</td>
			<td>used to quantify DNA plasmid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">water purificatiom unit</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">Elix 100</td>
			<td>use to make water type 1 (ASTM) used in spectrophotometry and DNA dilutions\</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">distilled water</td>
			<td style="width:227px;"></td>
			<td style="width:307px;">-</td>
			<td>used in gels, culture medium, stainings and general solutions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">HCl</td>
			<td style="width:227px;">J.T. Baker</td>
			<td>9535-03</td>
			<td>used to make non-denaturing polyacrylamide gel tris-HCl buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">6X DNA loading dye</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">R0610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">5 mm Blue LED</td>
			<td style="width:227px;">Jinyuan electronics</td>
			<td style="width:307px;">SP-021</td>
			<td>light source used in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">protoboard</td>
			<td style="width:227px;">KANDH</td>
			<td style="width:307px;">KH102</td>
			<td>light source support and circuit&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">9 V battery</td>
			<td style="width:227px;">Duracell</td>
			<td style="width:307px;">-</td>
			<td>used to power the LED&#39;s</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:187px;">horizontal electrophoresis chamber</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1704486EDU</td>
			<td>used to make and run agarose gel in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">vertical electrophoresis kit</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1658002EDU</td>
			<td>used to run polyacrylamide gels in calibration curves</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">power supply</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1645050&#160;</td>
			<td>used to power the electrophoresis</td>
		</tr>
	</tbody>
</table>

dna staining method based on formazan precipitation induced by blue light exposure

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the formation of a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in a solution of 1x (equivalent to 2.0 &#181;M) SYBR Green I (SG I) and 0.20 mM nitro blue tetrazolium that produces a purple precipitate of formazan when exposed to sunlight or specifically blue light. Also, DNA recovery tests were performed using an ampicillin resistant plasmid in an agarose gel stained with our method. A larger number of colonies was obtained with our method than with traditional staining using SG I with ultraviolet illumination. The described method is fast, specific, and non-toxic for DNA detection, allowing visualization of biomolecules to the "naked eye" without a transilluminator, and is inexpensive and appropriate for field use. For these reasons, our new DNA staining method has potential benefits to both research and industry.

A purple formazan precipitate appears where the DNA is located (verified by mass spectrometry13). In the experiment, a DNA ladder was loaded to make a calibration curve (Figure 3). The protocol works for both acrylamide and agarose gels, but it has a lower intensity and takes a longer time in agarose. However, the use of agarose gels allows the recovery of the sample using a commercially available kit, and it does not interfere with th...

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DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

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Research

JoVE Journal

Biochemistry

9.4K Views.  Universidad de Chile. This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

Video: DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...