We acknowledge Melissa Grunlan for helpful discussions on SAM formation. This research was supported in part by the Robert A. Welch Foundation (A-1585).

Preparation of reagents
Prepare PIPES buffer (80 mM PIPES, 1mM MgCl2, 1mM EGTA, adjusted to pH 6.8 with KOH). Aliquot and store glucose oxidase, catalase and glucose at -70&deg;C and taxol at -20&deg;C according to existing protocols.1,2 Aliquot and store tubulin at -70&deg;C according to the instructions included by the supplier.
Pattern Au microelectrodes using lithography
<ol>
<li>Cut silicon wafers into 1 cm &acute; 1.5 cm substrates using a scribe or wafer cutter. Clean the substrates by placing them in a beaker with enough acetone to cover them and sonicate for 5 minutes. Without allowing the substrates to dry, rinse them in fresh acetone, then immediately rinse in isopropanol and dry using nitrogen.</li>
<li>Fabricate Au microelectrodes on the clean substrates using standard, positive-tone photolithography or electron beam lithography, depending on the size of the features desired.3 When designing the pattern geometry, keep the diameter of each electrode pattern below 100 microns to ensure coverage by the PEG SAM (described in the next section). Include contact pads (~ 300 mm
2) positioned 300 to 500 mm from the pattern with a 1 mm line connecting the pad with the electrode (Fig. 1). Place the lithographic patterns near the middle of the substrate to allow space around the pattern for assembling the flow chamber. <img border="0" src="/files/ftp_upload/1390/1390fig1thumb.jpg" alt="figure 1" /> Figure 1. Sample geometry. Please <a href="https://www.jove.com/files/ftp_upload/1390/1390fig1.jpg">click here</a> to see a larger version of figure 1.</li>
<li>After development of the pattern in the resist layer, scratch a line in the resist from the contact pad to the edge of the substrate using tweezers (Fig. 1). Upon metal deposition, the scratch will allow electrical contact between the electrode and the edge of the substrate. A deposition chamber, preferably with two sources, is used to deposit the metal layer. The electrodes are formed by deposition of 2 nm of Cr (for adhesion) followed by 6 nm of Au, without breaking vacuum. A quartz crystal microbalance equipped inside the chamber can be used to measure the thickness of the films during deposition.</li>
</ol>
Prepare the SAM
<ol>
<li>Preheat and oven in a well-ventilated area to 75&deg;C. Prepare the silanization solution by adding 20 mL of toluene to a 250 mL glass beaker. Then add 1 mL PEG-silane (i.e. 5% v/v) to the toluene using a plastic pipette. Mix in the PEG-silane well by pipetting up and down 5 to 10 times and then stirring with the pipette for 30 seconds.</li>
<li>Place the patterned samples in the beaker with the patterned side facing upwards, making sure that they are completely submerged in the PEG-silane toluene solution and are evenly spaced across the bottom of the beaker. Place the beaker in the oven and bake 18 to 21 hours at 75&deg;C. While the SAM is forming, prepare the counterelectrode (see below) and assemble any additional components required for the remaining steps.</li>
<li>After 18-21 hours in the oven, the toluene should evaporate leaving the patterned samples in a viscous PEG-silane residue. Add 20 mL of toluene to the beaker and soak the samples for 1-2 minutes. Remove the samples from the beaker and rinse with toluene, then isopropanol, and finally dry with nitrogen. At this point, the samples should look clean and the SAM layer should be invisible to the naked eye. SAMs prepared this way are usable for up to two days when stored under cool dry conditions (25 &deg;C, moderate humidity). Unsuccessful SAM formation results in a cloudy or uneven hued residue coating on the surface.</li>
</ol>
Fabricate the counterelectrode
In the same deposition chamber setup used for microelectrode deposition, make a counterelectrode by depositing 1 nm of Cr, followed by 4 nm of Au onto a 22 x 50 mm No. 1 or No. 0 glass coverslip. The counterelectrode should be nearly transparent with a light gray or pink tint. Store the counterelectrode coated-side-up in a Petri dish to keep it clean.
Polymerize tubulin into MTs
<ol>
<li>Thaw unlabeled tubulin and rhodamine tubulin and immediately combine at a ratio of 1:2 labeled to unlabeled to obtain bright MTs. Mix by pipetting up and down several times. Polymerize the tubulin for 20-40 minutes in a heated water bath at 37&deg;C. The length of the MTs will be greater for longer polymerization times. While the tubulin is polymerizing, prepare a PIPES-taxol buffer by adding taxol to 20 mM into a microcentrifuge tube containing PIPES buffer. Mix the taxol well by pipetting up and down several times and vortexing, and then place the tube into the heated bath at 37&deg;C. After polymerization, dilute the MTs by 1/100 in warm PIPES-taxol buffer and shield from light. MTs prepared in this way will elongate over time and bundle, which can be reduced by further dilution.</li>
<li>Prepare a 10x oxygen scavenging mixture (OS) by combining 30 mL cold PIPES buffer (0-4&deg;C), 5 mL of 50% 2-mercaptoethanol, 5 mL glucose oxidase, 5 mL catalase, and 5 mL glucose.2 The glucose should be added last. Mix by pipetting up and down 3-5 times after adding each component. Place the OS mixture on ice; it will remain effective for about 2 hours.</li>
<li>Verify that the MT polymerization was successful and that the MTs are stable under several minutes of fluorescence excitation by imaging the prepared MTs with 1x OS mix. For imaging the MTs prepared in this way, be sure to use an excitation/emission filter combination suitable for rhodamine fluorescence. Determine the best working dilution for imaging individual filaments by further diluting the MTs by 1/10 to 1/100 using PIPES buffer and imaging with 1x OS mix.</li>
<li>Prepare a flow chamber by sandwiching a large (22 x 50 mm) and small (22 x 22 mm) glass coverslip with two pieces of double-sided tape placed 2-3 mm apart. The tape is easily cut into strips by placing it on a glass slide and cutting with a razor blade. Introduce a few microliters of MTs into the flow cell using a micropipette. For high magnification imaging, long working distance objectives may be required for imaging the upper surface of the flow chamber. The distance from the coverslip/sample surface to the upper surface of the flow chamber is determined by the thickness of the tape and should be 60-100 mm.</li>
</ol>
Pattern MTs using electrophoresis
<ol>
<li>Prepare an electrophoresis chamber by placing two thin strips of double-sided tape across the patterned, SAM-coated sample such that the electrode is between the strips with the connecting path running perpendicular to the tape (Fig. 2). Place the counterelectrode on the double-sided tape with the metal side touching the tape such that the edge of the sample overhangs the counterelectrode by about 3 mm (Fig. 2). Cut several 15 cm lengths of the insulated copper magnet wire and strip 1 cm of the insulation off of both ends. Attach a wire to the exposed Au area of the substrate using a small drop of silver paint while taking care to avoid making electrical contact with the counterelectrode (this can be checked with a multimeter). Attach a second wire to the counterelectrode using silver paint. Wires attached with silver paint can be further secured to the samples with tape. <img src="/files/ftp_upload/1390/1390fig2thumb.jpg" border="0" alt="figure 2" /> Figure 2. Flow cell assembly for an inverted fluorescence microscope. Please <a href="https://www.jove.com/files/ftp_upload/1390/1390fig2.jpg">click here</a> to see a larger version of figure 2.</li>
<li>Introduce MTs at the desired dilution using PIPES buffer combined with OS at 1x final concentration. Image at 20X magnification and locate the pattern on the silicon wafer before applying a voltage. Optionally, the flow chamber can be sealed using nail polish to prevent fluid flow and MT migration in the plane of the flow chamber.</li>
<li>Apply a voltage up to 1 V and observe migration of MTs. Lower voltages (down to about 0.5 V) may be used if reversible trapping of MTs is desired; however this requires sealing the flow chamber to prevent lateral drift. Nail polish works well for sealing the open ends of the flow chamber. At voltages above about 0.7 V, some MT adhesion may be seen on the pattern, though it will be weak. At voltages above about 0.9 V, the MT migration will be faster, and the adhesion will be stronger. Above about 1.2 V the likelihood for inducing electrolysis (gas bubble formation) and also for destroying the microelectrodes increases significantly. Migration can be tracked by adjusting the vertical plane of focus of the microscope. If carried out correctly, the MTs will localize at the electrode surfaces.</li>
</ol>

<ol>
	<li>Rai-Choudhury, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Microlithography, Micromachining, and Microfabrication: Microlithography. , (1997).</li></ol>

The authors have nothing to disclose.

The migration of MTs is easily visualized using fluorescence microscopy because of their brightness and the low background fluorescence. The method is generally applicable to biomolecule patterning, as it only requires that the biomolecules be induced to carry a net charge by suitable adjustment of the buffer pH. Electroosmosis is avoided in this setup because there are no charged surfaces such as the silica walls used in capillary electrophoresis.
Several modifications of this method are pos...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2-[methoxypoly-(ethyleneoxy)propyl]-trimethoxysilane (PEG-silane)</td>
<td>&nbsp;</td>
<td>Gelest, Inc.</td>
<td>SIM6492.7</td>
<td>6-9 PEG units;
molecular weight 450-600 g mol-1</td>
</tr>
<tr>
<td>2-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>Acetone</td>
<td>&nbsp;</td>
<td>EMD Chemicals</td>
<td>AX0120-8</td>
<td>ACS grade</td>
</tr>
<tr>
<td>Catalase</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>219001-5MU</td>
<td>&nbsp;</td>
<tr>
<td>Chlorobenzene</td>
<td>&nbsp;</td>
<td>EMD Chemicals</td>
<td>MK441908</td>
<td>ACS grade</td>
</tr>
<tr>
<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#x2019;,N&#x2019;-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>324626</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>EMD Chemicals</td>
<td>DX0145-1</td>
<td>&nbsp;</td>
<tr>
<td>Glucose oxidase</td>
<td>&nbsp;</td>
<td>MP Biomedicals</td>
<td>100330</td>
<td>&nbsp;</td>
<tr>
<td>Guanosine-5&#x2019;-[(&alpha;,&Beta;)-methyleno]triphosphate, Sodium salt (GMPCPP)</td>
<td>&nbsp;</td>
<td>Jena Biosciences, GmbH</td>
<td>NU-405s</td>
<td>&nbsp;</td>
<tr>
<td>Guanosine-5&#x2019;-triphosphate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8752</td>
<td>&nbsp;</td>
<tr>
<td>Isopropanol</td>
<td>&nbsp;</td>
<td>EMD Chemicals</td>
<td>PX1835-5</td>
<td>ACS grade</td>
</tr>
<tr>
<td>Methyl isobutyl ketone (MIBK)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>AC12739-0010</td>
<td>&nbsp;</td>
<tr>
<td>Paclitaxel (taxol)</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>580555</td>
<td>&nbsp;</td>
<tr>
<td>Piperazine-N,N''-bis(2-ethanesulfonic acid) (PIPES)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1851</td>
<td>&nbsp;</td>
<tr>
<td>Polymethylmethacrylate (PMMA)</td>
<td>&nbsp;</td>
<td>Brewer Science, Inc.</td>
<td>950K</td>
<td>molecular weight 950K</td>
</tr>
<tr>
<td>Rhodamine tubulin</td>
<td>&nbsp;</td>
<td>Cytoskeleton, Inc.</td>
<td>T331M</td>
<td>from bovine brain 99% pure, lyophilized</td>
</tr>
<tr>
<td>Toluene</td>
<td>&nbsp;</td>
<td>EMD Chemicals</td>
<td>TX0735-5</td>
<td>ACS grade</td>
</tr>
<tr>
<td>Tubulin</td>
<td>&nbsp;</td>
<td>Cytoskeleton, Inc.</td>
<td>T238</td>
<td>from bovine brain 99% pure, lyophilized</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
			No. 1 borosilicate glass coverslips (22 X 50 mm) were purchased from VWR. Polished electronic grade p-type &lt;111&gt; silicon wafers were purchased from Addison Engineering, Inc. High-purity silver paint was obtained from Structure Probe, Inc. (Catalog # 5001 AB). Insulated fine gauge copper magnet wire (~ 0.08 mm (0.003&#148;) diameter) can be obtained from electronic supply companies.		</div>

antifouling self-assembled monolayers on microelectrodes for patterning biomolecules

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single assembly step and prevents biofouling on silicon and gold surfaces. The SAM is used to coat microelectrodes patterned with standard, positive-tone lithography. Using the microtubule as an example, we apply a DC voltage to induce electrophoretic migration to the SAM-coated electrode in a reversible manner. A flow chamber is used for imaging the electrophoretic migration and microtubule patterning in situ using epifluorescence microscopy. This method is generally applicable to biomolecule patterning, as it employs electrophoresis to immobilize target molecules and thus does not require specific molecular interactions. Further, it avoids problems encountered when attempting to pattern the SAM molecules directly using lithographic techniques. The compatibility with electron beam lithography allows this method to be used to pattern biomolecules at the nanoscale.

Watch this Scientific Journal Video about Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules at JoVE.com

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

We present a procedure for forming a poly(ethylene glycol) self-assembled monolayer (PEG-SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single step and prevents biofouling on silicon and gold surfaces. Electrophoresis is then used for patterning biomolecules down to the nanoscale.

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold ...

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11.4K Views.  Texas A&M University (TAMU). We present a procedure for forming a poly(ethylene glycol) self-assembled monolayer (PEG-SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single step and prevents biofouling on silicon and gold surfaces. Electrophoresis is then used for patterning biomolecules down to the nanoscale.

Video: Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

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

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

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

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

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...