Funding for the development of the extraction pipette was provided in part by an Internal Research and Development grant awarded to DRP.

1. Sample Lysis
<ol>
 <li>Samples should be in liquid form. If sample is solid, for example food or stool, the sample should be suspended in a liquid media such as water or phosphate buffered saline (PBS).</li>
 <li>Mix 500 &mu;L of the sample with 500 &mu;L of 6M Guanidine Thiocyanate (pH 6.5) in a 1.5 mL tube.</li>
 <li>Depress the bulb of an extraction pipette, expelling the ambient air.</li>
 <li>Pull the entire 1 mL sample into the extraction pipette, over the sorbent material, by allowing the bulb to slowly expand.</li>
 <li>Invert the extraction tool such that the sorbent beads and sample drain into the bulb.</li>
 <li>Grind the sorbent beads with the sample in the bulb being careful not to expel the sample out of the extraction pipette.</li>
</ol>
2. Nucleic Acid Extraction (Figure 1, Panel A)
<ol>
 <li>Invert the extraction pipette (opening downward) and expel the sample into the original 1.5 mL tube.</li>
 <li>Pull the entire 1 mL sample into the extraction pipette, passing over the sorbent, at a moderate rate, being careful to keep the sorbent in the neck of the extraction pipette.</li>
 <li>Expel the entire sample into the 1.5 mL tube by depressing the bulb.</li>
 <li>Repeat steps 2.2 and 2.3, passing the sample over the sorbent four more times for a total of 5 passes.</li>
 <li>After the fifth pass over the sorbent, expel the entire 1 mL sample into 4mL of the protein extraction buffer (in a 15 mL conical tube), close the tube, and set aside for later use.</li>
 <li>Wash the isolated nucleic acids by passing 1 mL of 95% ethanol over the sorbent three times, returning the ethanol to its original tube upon final passage.</li>
 <li>Repeat step 2.6 using a second 1 mL, 95% ethanol wash.</li>
 <li>Press and release the bulb for 5 minutes, passing ambient air throughout the extraction pipette to dry the nucleic acids/sorbent bed. Wipe residual ethanol from the tip of the extraction pipette using a Kimwipe.</li>
 <li>Recover the nucleic acids by passing 250 &mu;L of 10 mM Tris (pH 6.8) over the sorbent five times. The Tris buffer can be replaced by any other suitable aqueous solution such as PBS. The resulting solution contains the extracted nucleic acids.</li>
</ol>
3. Protein Extraction (Figure 1, Panel B)
<ol>
 <li>Mix the sample and protein extraction buffer tube from step 2.5 by inversion and pass approximately two to three milliliters of this 5 mL sample over the sorbent of a new extraction pipette.</li>
 <li>Expel the entire sample into the same 15mL conical tube being careful to keep the sorbent in the neck of the extraction pipette.</li>
 <li>Repeat steps 3.1 and 3.2, passing the sample over the sorbent 15 times.</li>
 <li>Wash the sorbent and the bound protein by passing 1 mL of 95% ethanol over the sorbent bed three times, ejecting the ethanol into it's original tube.</li>
 <li>Repeat step 3.4 using a second 1 mL, 95% ethanol wash.</li>
 <li>Press and release the bulb for 5 minutes, passing ambient air throughout the extraction pipette to dry the protein/sorbent bed. Wipe residual ethanol from the tip of the extraction pipette using a kimwipe.</li>
 <li>Recover the protein by passing 250 &mu;L of PBS over the sorbent five times. The PBS buffer can be replaced by any other suitable aqueous solution such as 10 mM Tris (pH 6.8). The resulting solution contains the protein content of the sample.</li>
</ol>
<img src="/files/ftp_upload/4202/4202fig1.jpg" alt="Figure 1A" /> 
Figure 1. 
<img src="/files/ftp_upload/4202/4202fig2.jpg" alt="Figure 2" /> 
Figure 2. 
<img src="/files/ftp_upload/4202/4202fig3.jpg" alt="Figure 3" /> 
Figure 3. 
<img src="/files/ftp_upload/4202/4202fig4.jpg" alt="Figure 4" /> 
Figure 4. 
<img src="/files/ftp_upload/4202/4202fig5.jpg" alt="Figure 5" /> 
Figure 5.

<ol>
	<li>Gupta, S. K., Keck, J., Ram, P. K., Crump, J. A., Miller, M. A., Mintz, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+data+gaps+pertaining+to+enterotoxigenic+Escherichia+coli+infections+in+low+and+medium+human+development+index+countries,+1984-2005.">Analysis of data gaps pertaining to enterotoxigenic Escherichia coli infections in low and medium human development index countries, 1984-2005.</a> Epidemiology and Infection. 136, 721-738 (2007).</li><li>Pawlowski, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+Pipette.">Extraction Pipette.</a> United States Patent and Trademark Office. , (2010).</li><li>Boom, R., Sol, C. J., Salimans, M. M., Jansen, C. L., Wertheim-van Dillen, P. M., van der Noordaa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+simple+method+for+purification+of+nucleic+acids.">Rapid and simple method for purification of nucleic acids.</a> J. Clin. Microbiol. 28, 495-503 (1990).</li><li>Karalus, R. J., Pawlowski, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+and+Device+for+Isolating+a+Protein+Sample.">Method and Device for Isolating a Protein Sample.</a> United States Patent and Trademark Office. , (2010).</li><li>Huang, A., Friesen, J., Brunton, 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=Characterization+of+a+bacteriophage+that+carries+the+genes+for+production+of+Shiga-like+toxin+1+in+Escherichia+coli.">Characterization of a bacteriophage that carries the genes for production of Shiga-like toxin 1 in Escherichia coli.</a> Journal of Bacteriology. 169, 4308-4312 (1987).</li><li>O'Brien, A. D., Newland, J. W., Miller, S. F., Holmes, R. K., Smith, H. W., Formal, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shiga-like+toxin-converting+phages+from+Escherichia+coli+strains+that+cause+hemorrhagic+colitis+or+infantile+diarrhea.">Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea.</a> Science. 226, 694-696 (1984).</li><li>Wagner, P. L., Neely, M. N., Zhang, X., Acheson, D. W. K., Waldor, M. K., Friedman, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+a+Phage+Promoter+in+Shiga+Toxin+2+Expression+from+a+Pathogenic+Escherichia+coli+Strain.">Role for a Phage Promoter in Shiga Toxin 2 Expression from a Pathogenic Escherichia coli Strain.</a> Journal of Bacteriology. 183, 2081-2085 (2001).</li><li>Mizutani, S., Nakazono, N., Sugino, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+So-called+Chromosomal+Verotoxin+Genes+are+Actually+Carried+by+Defective+Prophages.">The So-called Chromosomal Verotoxin Genes are Actually Carried by Defective Prophages.</a> DNA Research. 6, 141-143 (1999).</li><li>Ptashne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Genetic Switch: Phage Lambda Revisited. , (2004).</li><li>Sambrook, J., Russell, D. W. <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. , (2001).</li><li>Feng, P., Monday, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+PCR+for+detection+of+trait+and+virulence+factors+in+enterohemorrhagic+Escherichia+coli+serotypes.">Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes.</a> Mol. Cell. Probes. 14, 333-337 (2000).</li></ol>

Production and Free Access to this article is sponsored by the United States Defense Threat Reduction Agency.

The tool, chemistries and protocol described in this report represent the first known example of an electricity-free, multi-macromolecule extraction system that can be utilized in field-based, point-of-care applications. Both macromolecule types can be applied to a number of downstream diagnostic or analytical devices. For example, the isolated nucleic acids are of PCR quality (Figure 2 and 3) while the protein component of the sample is immunoreactive (Figure 4). The utilization of this extraction system in austere or resource limited areas of the world could drastically reduce the burden of disease on the populations living there. This extraction system could also aid in disease surveillance programs throughout the world by providing a robust yet rapid and cost effective method for sample processing in austere or remote locations.
One important aspect to the extraction method outlined above is that it can be modified by the user. For example, in cases where sample size is greater or less than the initial 500 &mu;Ls (step 1.2), the user can adjust the volumes of the reagents accordingly. That is, the volumetric ratio of the reagents to the sample size should remain constant while the absolute volumes can be altered. Also, adjustments in the number of passages over the sorbent can also be made at the user's discretion. For example, if a larger sample volume is used, passing the sample over the sorbent more times than listed (steps 2.4 &amp; 3.3) will increase the likelihood of the macromolecule type (nucleic acid or protein) contacting the sorbent. The increase in passages should increase capture efficiency until sorbent saturation is reached.
We have found that the extraction tool and associated chemistries have some limitations, the most significant of which is that the protein extraction chemistry is inefficient for the recovery of highly glycosylated proteins. In the event that the downstream diagnostic targets a glycosylated protein, this system may not be ideal for identification. Additionally, the upper and lower limits of extraction efficiencies for either nucleic acids or proteins are still being determined, and optimization is underway to maximize recovery efficiencies. Further development of the tool will overcome these current knowledge gaps.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company </td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Guanidine Thiocyanate</td>
			<td>Teknova</td>
			<td>G4000</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Pharmco-Aaper</td>
			<td>11ACS200</td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Sigma-Aldrich</td>
			<td>109827-1L</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-4503</td>
		</tr>
	</tbody>
</table>

electricity-free, sequential nucleic acid and protein isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

Nucleic Acid Isolation: Isolated nucleic acids are PCR amplifiable and free of protein contamination.
Example 1: The recovered nucleic acids are suitable for use in PCR-based applications. For example, Figure 2 shows the amplification of shiga toxin genes associated with Enterohemorrhagic E. coli O157:H7 strain EDL-933 (ATCC # 35150) from a nucleic acid/protein extraction procedure using the extraction pipette. The shiga toxin genes stx1 and stx2 are found on temperate lambda-like bacteriophage and defective bacteriophage in this and other Enterohemorrhagic E. coli strains under the control of the PR promoter 5 6 7 8. The activation of the SOS response in E. coli leads to prophage induction and/or toxin production 9. We've utilized a modified version of the multiplexed PCR assay by Feng et al.11 to identify the stx1 and stx2 genes in both culture and spiked sewage samples. Lanes A and B in Figure 2 show the results of a PCR amplification of an unprocessed culture sample (lane A) and a processed culture sample (lane B). These results are virtually identical meaning there is no loss of fidelity in the processed or isolated nucleic acids. We next amplified spiked raw sewage (lane D) or isolated nucleic acids from spiked sewage (lanes E &amp; F). These data indicate that the isolated nucleic acids resulted in greater amplification versus the non-isolated nucleic acids from spiked sewage samples. We therefore conclude that either PCR inhibitors naturally found in sewage were removed upon isolation or that the nucleic acids were concentrated upon isolation. A combination of these two hypotheses is also possible.
Ultraviolet spectroscopy was next used to show that the isolated nucleic acids were relatively free of contaminating protein. Figure 3 shows the UV spectra of two E. coli O157:H7 nucleic acid extractions, labeled isolation a and isolation b, in comparison to the normalized spectrum of the bacterial culture, labeled pre-isolation. The UV spectra of the isolated nucleic acids resembles the spectra of pure nucleic acids, having a calculated ratio of absorbance between 260 nm and 280 nm approaching 1.810. These data show that there is very little contaminating protein in the nucleic acid fraction recovered using the technique described.
Protein Isolation: Recovered protein from the nucleic acid/protein isolation experiment is immunoreactive and electrophoretically identical to pure protein.
Example 2: The isolated protein fraction was applied to the RapidChek E. coli O157 lateral flow assay strips (SDIX, Inc.). Controls show a positive identification in all samples harboring E. coli O157, with or without STX induction (Figure 4, lanes B and C, respectively). The data in Figure 4 also show positive results for those samples harboring E. coli O157 that were processed for nucleic acids and then protein using the extraction pipette. These data show that the isolated protein from each sample is immunoreactive thus triggering a positive response.
PCR amplification (as described in Example 1) was performed on the protein samples to show that the isolated protein fraction was devoid of contaminating nucleic acids. The results for these experiments were all negative by gel electrophoresis. Thus, we conclude that the protein content of the sample was separated from contaminating nucleic acids. These results, taken in whole with those described for the nucleic acid isolation step show that the nucleic acids and protein content are isolated in separate fractionation steps.
Example 3: The isolated protein is suitable for electrophoresis 10. Figure 5 depicts a coomassie stained 8% acrylamide gel loaded with BSA (Figure 5, lanes A &amp; C) and BSA isolated from 6M guanidine isothiocyanate using the extraction pipette and associated protocol. The electrophoretic mobility of the isolated protein is identical to that of the control samples. We thus conclude that the extraction process does not alter the covalent structure of the isolated protein.
<img alt="Figure 1a" src="/files/ftp_upload/4202/4202fig1A.jpg" /> 
Figure 1, Panel A: Depiction of a typical nucleic acid extraction procedure. The sample is mixed with 6M guanidine thiocyanate in the sample tube and passed over the sorbent five times. After the last passage, the sample is added to the protein extraction buffer. The sorbent and bound nucleic acids are washed twice with ethanol. The sample is then air dried and eluted from the sorbent.
<img alt="Figure 1b" src="/files/ftp_upload/4202/4202fig1B.jpg" /> 
Figure 1, Panel B: Depiction of a typical protein extraction procedure. The sample mixed with protein isolation buffer from step 1, Figure 1, panel A is passed over the sorbent of a second extraction pipette 15 times. The remainder of the procedure is identical to that of the nucleic acid extraction protocol.
<img alt="Figure 2" src="/files/ftp_upload/4202/4202fig2.jpg" /> 
Figure 2: Negative picture of an ethidium bromide stained agarose gel. A multiplexed shiga toxin 1 and 2 gene amplification experiment was performed using culture samples (lanes A and B) or raw sewage samples spiked with E. coli O157:H7 (lanes D, E and F). The samples were either directly added to a PCR reaction tube (lanes A and D) or the samples were processed using the isolation process and the extracted nucleic acids were added to the PCR reaction tube (Samples B, E and F). The PCR protocol is a modification of the one described by Feng et al11. Our modification targeted only those amplified by the stx1 (lower band) and stx2 (upper band) primer pairs, dropping out all other primer pairs. Lane C is the 1Kb ladder from BioRad.
<img alt="Figure 3" src="/files/ftp_upload/4202/4202fig3.jpg" /> 
Figure 3: UV spectroscopy results of E. coli O157:H7 nucleic acids isolated using the reported extraction pipette. The isolated nucleic acids are labeled isolation a and isolation b. The UV spectrum of the sample was also tested prior to nucleic acid extraction and is labeled pre-isolation. The spectra were obtained using a Hitachi U3010 Spectrophotometer and associated software package.
<img alt="Figure 4" src="/files/ftp_upload/4202/4202fig4.jpg" /> 
Figure 4: Color photo of RapidChek test strips. The protein content of the samples used in Figure 2 was isolated using the extraction pipette and tested for immuno-reactivity by lateral flow assay. Lanes A-C are unprocessed controls, that is, cell culture was added to the test strip without nucleic acid or protein extraction. Lane A is E. coli BL21 DE3 pLysS as a negative control, Lanes B and C show E. coli O157:H7 that was untreated (B), or treated with mitomycin C for 4 hours (C). Lanes D through H show the results of adding the extracted protein to the lateral flow test strips. Lanes D and E were probed using protein extract from E. coli BL21 DE3 pLysS either without (D) or with mitomycin C treatment (E) as negative controls. Lanes F-H show the results when extracted protein from E. coli O157:H7 was used. Protein was extracted from untreated (F), or from 2 hour (G) or 4 hour (H) Mitomycin C treated cultures. Positive results produce a double red line.
<img alt="Figure 5" src="/files/ftp_upload/4202/4202fig5.jpg" /> 
Figure 5: Photograph of a Coomassie stained SDS-PAGE. BSA was isolated from solution using the extraction pipette and described protocols. Lanes A and C are control lanes of 250 &mu;g/mL and 500 &mu;g/mL respectively. Lanes B and D depict recovered BSA from solutions containing the same concentrations as their respective controls.

Watch this Scientific Journal Video about Electricity-Free, Sequential Nucleic Acid and Protein Isolation at JoVE.com

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

electricity-free-sequential-nucleic-acid-and-protein-isolation

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12.4K Views.  CUBRC, Inc.. A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

Video: Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

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 ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...