This work was supported by the National Institute on Aging (AG039768) to MHK. We thank Drs. Thomas Sharkey and Honggao Yan for generously providing the instruments, as well Sean Weise and Yan Wu for technical assistance.

1. Preparation of Reagents
<ol>
	<li>Prepare aggregation buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA). Store at RT, stable for months. Supplement 1 mM dithiothreitol (DTT) before use. 
	NOTE: a HEPES-based buffer (10 mM HEPES, pH 7.5, 0.1 mM EDTA, 5 mM DTT) also produces similar results in tau aggregation.</li>
	<li>Prepare thioflavin T or thioflavin S stock solution (3 mM, dissolved in aggregation buffer), and filter by 0.22 &#956;m sterile filter unit. Store at &#8211;20 &#176;C in a tube covered by aluminum foil, stable for months.</li>
	<li>Prepare heparin stock solution (300 &#956;M, dissolved in aggregation buffer). Store at &#8211;20 &#176;C, stable for months.</li>
	<li>Prepare dithiothreitol (DTT) stock (1 M, dissolved in water). Aliquot into 1.5 ml tubes. Store at &#8211;20 &#176;C. Before aggregation assays, thaw the 1 M solution at RT. From this 1,000x stock, prepare an aliquot of 100 mM working stock with deionized water. Leave on ice until ready.</li>
	<li>Remove tau from &#8211;80 &#176;C freezer. Thaw on ice. Adjust tau to predetermined concentration with the aggregation buffer. Spin in a microcentrifuge at 20,800 x g for 10 min at 4 &#176;C to remove pre-formed large aggregates. This pre-spinning step increases consistency in the subsequent fluorescence measurement of each batch of the protein prep. Transfer the supernatant to another tube; leave on ice until ready to assemble the aggregation reaction.</li>
</ol>
2. No-dye, Terminal Assay
NOTE: The aggregation reaction of this assay is done in the absence of the fluorescent dye. After mixing all components, the reaction is allowed to proceed to pre-determined time points. Aliquots are then taken out of the aggregation reaction and mixed with ThT or ThS for amyloid binding before fluorescence reading. The initial volume of the aggregation reaction depends on the number of time points needed. This approach may require a large amount of tau protein, but is fast, straightforward, and can be done in a fluorometer or a multi-well plate reader (see Discussion). Below is the step-by-step operation, using the ISA SPEX FluoroMax-2 compact spectrofluorometer for fluorescence quantification.
<ol>
	<li>Set up the aggregation mixture in 1.5 ml Eppendorf tubes as in Table 1. Each column represents the ingredients needed for a 100 &#956;l reaction, which is enough for one time-point measurement. Adjust the amount for the entire aggregation mix based on the time points needed for the particular experiment. Add additional 10% of each component to give room for pipetting error. The typical reaction containing heparin shown here can be replaced by arachidonic acid or aggregation buffer. Add DTT to 1 mM to the reaction mixture. If the entire reaction lasts more than one day, supplement fresh DTT everyday (1 mM) to ensure a reducing environment.</li>
	<li>Invert the tube a few times to mix. Place each reaction in a 37 &#176;C incubator or water bath. Agitation is not needed for tau aggregation.</li>
	<li>Before measuring the fluorescence, turn on the spectrofluorometer (lamp first, then computer). 
	NOTE: The xenon arc lamp that can be used right away. However, for best results allow the machine to warm up for about 10 min before reading fluorescence.</li>
	<li>Start the software on the computer.
	<ol>
		<li>Choose Real Time Display mode in Instrument Control Center, set excitation wavelength to 450 nm (slit to 2 nm) and emission wavelength to 510 nm (slit to 5 nm). Close Real Time Display mode window to return to the Instrument Control Center.</li>
		<li>Choose Constant Wavelength Analysis, press Add&#62;&#62; key in the upper frame to add wavelengths sets. Set acquisition parameters of Standard Error to 1 and Maximum Trials to 3, then click Add. Click Go! to open the Data Display window.</li>
		<li>In the Data Display window, click Start Acq to open the New Sample dialog box. Choose &#8220;unknown&#8221; for sample type.</li>
	</ol>
	</li>
	<li>To every 100 &#956;l aggregation mixture, add 98 &#181;l aggregation buffer and 2 &#181;l 3 mM thioflavin T. Pipet several times to mix.</li>
	<li>Transfer the entire mixture to a cuvette (FCA3, exterior dimension, w x l x h = 12.5 mm x 12.5 mm x 45 mm). Place the cuvette in the sample holder in the sample-compartment and close the lid. Click Run to collect the fluorescence data. Record the data.</li>
	<li>Remove the cuvette and decant the solution. Rinse the cuvette by distilled water 3 times. Dry by blowing air in and outside the cuvette.</li>
</ol>
3. With-dye, Continual Mode Assay on a SpectraMax M2 Plate Reader
NOTE: This assay differs from the previous one in that the fluorescent dye ThT or ThS is included in the aggregation reaction. This allows continuous measurement of the same set of reactions. Because of the repetitive use of the reaction, this method is better done with an automatic multi-well plate reader (as shown below the operation of SpectraMax M2). A regular fluorometer also works but the frequency of measurement of fast aggregation reactions is somewhat limited, due to the manual nature of the operation.
<ol>
	<li>Set up the aggregation mix in a 96-well plate (96-well black solid plate, well-volume 360 &#956;l, flat bottom) as in Table 2. Each column represents the ingredients needed for a 200 &#956;l reaction, which is enough for one time-point measurement. Mix well by pipetting several times. Supplement fresh 1mM DTT every day throughout the course of experiments.</li>
	<li>Incubate the 96-well plate at 37 &#176;C.</li>
	<li>At each time point before fluorescence measurement, turn on the multi-mode microplate reader and the computer. Allow sufficient time for the machine to stabilize, about 10 min.</li>
	<li>Start the software on the computer. Set temperature to 37 &#176;C and choose Fluorescence Intensity (FI-Top Read) mode, set excitation wavelength at 450 nm and emission wavelength at 510 nm.</li>
	<li>Insert the 96-well plate into the drawer and press the READ key to start measurement.</li>
	<li>After reading, remove the plate and return it back to the 37 &#176;C incubator. Copy the data and paste into an Excel spreadsheet for data analysis and plotting.</li>
</ol>
4. With-dye, Continual Mode Assay on an Compact Spectrofluorometer
<ol>
	<li>Set up the aggregation mixture in 1.5 ml Eppendorf tubes as in Table 3. Each column represents the ingredients needed for a 200 &#956;l reaction, which is enough for one time-point measurement.</li>
	<li>Invert the tube a few times to mix.</li>
	<li>Turn on the spectrofluorometer and set the software as in steps 2.3 and 2.4.</li>
	<li>Transfer the entire mixture to a cuvette. Place the cuvette in the sample holder in the sample-compartment and close the lid. Click Run to collect the fluorescence data. Record the data.</li>
	<li>Continue reading at appropriate intervals by clicking Run and recording the data. If the aggregation is to be monitored at a high frequency (e.g., every 30 or 60 sec), leave the reaction in the cuvette and in the machine until either the measurement is finished, or when there is sufficient time to swap reactions or cuvettes.</li>
	<li>Remove the cuvette and decant the solution. Rinse the cuvette by distilled water 3 times. Dry by blowing air in and outside the cuvette.</li>
</ol>

We have nothing to disclose.

This protocol demonstrates different assay conditions and instruments that detect the phosphorylation-dependent fast tau aggregation kinetics. In the terminal assay, the fluorescence dye ThT is added to a portion of the reaction removed from the master mix at each time point. Amyloid binding-induced fluorescence is then measured 26. In the second, with-dye mode, tau aggregation is carried out in the presence of ThT or ThS, rendering this type of reaction suitable for real-time automatic assessment of the growt...

Alzheimer&#8217;s disease (AD) is one of a large collection of neurodegenerative disorders known as tauopathies. The quintessential pathology underlying tauopathy is the neurofibrillary tangles, NFTs, in neurons, astrocytes and microglia 1-4. The NFT density correlates with cognitive impairment 3,5 and neuron loss 6. NFT contains primarily hyperphosphorylated tau protein (referred to as &#8220;p-tau&#8221; henceforth) that forms straight or paired helical filaments (PHF) 7,8. Tau is a microtubule associated protein thought to facilitate axonal transport that is essential for neuronal signaling and trafficking 9,10. Each tau molecule contains 2 to 3 phosphates in normal brain, but the phosphoryl content increases by several folds in tauopathy patients 11. Multiple kinases are likely to contribute to tau hyperphosphorylation including GSK3&#946; (glycogen synthase kinase 3&#946;) and CDK5 (cyclin-dependent kinase 5) 12,13, but the direct trigger for the pathological phosphorylation remains elusive 14. Abnormal phosphorylation in or near the microtubule-binding motifs dissociates tau from the microtubule 15, and causes tau mis-localization to the somatodendritic compartment, where p-tau oligomerizes into straight or paired helical filaments that can eventually polymerize into NFT inclusions. The close tie between tau hyperphosphorylation, NFT formation, and neurodegeneration led to a prevalent hypothesis that p-tau tangles elicit apoptotic and other cytotoxic responses, and thus is the underlying cause for tauopathy neurodegeneration 16,17. Drug screens and early clinical tests based on this premise have been launched 18. However, this hypothesis faces challenges 19,20. For example, SantaCruz et al. showed that cognitive functions of transgenic mice can be improved by suppressing the expression of a mutant human tau, even though NFTs continued to form from existing tau molecules 21. In a Drosophila model, NFT was shown to sequester the toxic cytosolic tau to protect the underlying neuron cells 22,23. Clearly, the pathogenesis role of NFT, if any, will greatly impact the direction of tauopathy therapeutics development.
In high concentrations, recombinant or normal brain tau protein spontaneously but slowly polymerizes into a PHF-like structure in vitro, as indicated by the binding of several &#946;-sheet preferred fluorescent dyes, electron microscopy, and light scattering spectroscopy 24-27. Adding heparin or arachidonic acid, an abundant fatty acid in human brain, drastically accelerates PHF formation in tau isoform- and inducer concentration-dependent manners 28-32. Intriguingly, hyperphosphorylated tau purified from AD brains or prepared by exhaustive in vitro phosphorylation reactions aggregates faster and more efficiently 26,33-35. These results are in excellent agreement with the pathological roles of p-tau. An in vitro system based on the aggregation of p-tau may thus serve as a powerful tool for AD drug screening.
Given the close association between tau aggregation and the progressive neurodegeneration of AD, as well as the recent failure in drug development targeting the A&#946; plaque, another key histological marker of AD 36-38, the interest in discovering drugs that control tau aggregation is rising. Indeed, several groups have already begun drug screens at different throughput, using in vitro tau aggregation reactions as the primary assay. A number of chemicals were found to exhibit inhibitory or reversal activities on tau aggregation in vitro 39-42. However, all current tau aggregation regulator screens use unmodified tau that misses the key pathological mark of phosphorylation, raising a concern for the specificity and efficacy of using these compounds in AD treatment.
One of the major hurdles of developing aggregation assays for biochemical characterization and AD drug screening is the production of sufficient quantities of the pathophysiologically relevant hyperphosphorylated tau protein. Using the Zippers-Assisted Catalysis system in which the 1N4R isoform of tau and the GSK-3&#946; kinase are co-expressed in E. coli as leucine zipper fusion proteins, we have overcome this challenge (Sui et al., submitted; see Figure 1 for the final products of tau and p-tau; also see 43 for preliminary mass spectrometry characterization of p-tau). From a panel of nine antibodies specific for different phosphorylation sites of tau, positive signals were seen in eight positions (data not shown). Below, we describe protocols and instrumentations that can differentiate the aggregation kinetic differences between unmodified tau and p-tau species. These assays were modified from published protocols that measured the increase of fluorescence of thioflavin T (ThT) or thioflavin S (ThS) upon amyloid (tau aggregates) binding 26. In the first &#8220;terminal&#8221;, no-dye approach, aggregation reactions are assembled and incubated in the absence of the amyloid dye. At different time points, an aliquot of each reaction is removed and mixed with equal volume of the ThT-containing buffer to stop aggregation and allow ThT to bind tau aggregates. Fluorescence is measured by an IAP FluoroMax-2 fluorometer. In the second &#8220;with-dye&#8221; continual monitoring assay, ThT or ThS is included in the aggregation reactions. Fluorescence can be measured continuously throughout the entire experiment manually or using a multi-plate reader. In addition, we describe an assay that uses a near-physiological concentration of tau and p-tau for aggregation in the continual measurement mode. The effect of phosphorylation remains readily detectable. Below, we will describe step-by-step operation procedures, and show representative results of these assays. Discussion of some of the pros and cons of each approach, as well as potential drug screening applications will follow.
At a high concentration, tau aggregates into amyloid-like structures spontaneously. However, in the laboratory, tau fibrillization is typically accelerated by such inducers as heparin (average molecular weight, 6,000 g/mol) and arachidonic acid. Examples shown herein include 30 &#181;M heparin. The formation of tau amyloid aggregates is monitored by the fluorescence resulting from amyloid binding by thioflavin T (ThT) or thioflavin S (ThS). Upon binding to tau aggregates, ThT exhibits a red shift in fluorescence (excitation: 450 nm; peak emission: 485 nm). ThS, on the other hand, has weak emission at 510 nm (excitation at 450 nm) before amyloid binding, but this fluorescence increases significantly in the presence of an amyloid protein such as the aggregated tau 44. Both dyes work well in detecting tau and p-tau aggregation. Because of the strong and relatively broad emission peak of ThT (see Figure 2), there is only 30% reduction in the fluorescence unit at 510 nm. For convenience, we use the same combination of excitation/emission wavelengths (i.e., 450 nm/510 nm) to monitor tau aggregation when using either dye.
Tau aggregation can be done in the presence or absence of the dye, depending on the purpose of the assay and the availability of tau protein. Both modes of reactions are shown below. In addition, we demonstrate the operation of two different instruments &#8211; a single-sample fluorometer (ISA-SPEX FluoroMax-2) and a multi-plate reader (SpectraMax M2). Readers should be able to adapt these protocols to suit their specific needs and instrument availability.

<table border="0" cellpadding="0" cellspacing="0" width="449">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:116px;">Name of the reagent</td>
			<td style="width:128px;">Company</td>
			<td style="width:103px;">Catalogue number</td>
			<td style="width:103px;">Comments</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:116px;">Trizma&#160;base</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:103px;">T1503</td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:116px;">NaCl</td>
			<td style="width:128px;">Macron Fine Chemicals</td>
			<td style="width:103px;">MAL-7581-06</td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:116px;">Ethylenediaminetetraacetic Acid (EDTA)</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:103px;">15576-028</td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:116px;">Thioflavin T</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:103px;">T3516</td>
			<td style="width:103px;">Stored in dark</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:116px;">Thioflavin S</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:103px;">T1892</td>
			<td style="width:103px;">Stored in dark</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:116px;">heparin</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:103px;">H3393</td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:116px;">DL-Dithiothreitol (DTT)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:103px;">D9779</td>
			<td style="width:103px;">Stored at 4&#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:116px;">96-well plate</td>
			<td style="width:128px;">Corning</td>
			<td style="width:103px;">3917</td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:116px;">ISA SPEX FluoroMax-2</td>
			<td style="width:128px;">Horiba</td>
			<td style="width:103px;"></td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:116px;">SpectraMax M2 Multi-Mode Microlate Reader</td>
			<td style="width:128px;">Molecular Devices</td>
			<td style="width:103px;"></td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:116px;">Mouse Anti-Tau Monoclonal Antibody</td>
			<td style="width:128px;">R&#38;D Systems</td>
			<td style="width:103px;">MAB3494</td>
			<td style="width:103px;">Stored at &#8211;80&#176;</td>
		</tr>
	</tbody>
</table>

in vitro aggregation assays using hyperphosphorylated tau protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

Using recombinant tau and p-tau (Figure 1), we established two different protocols to compare the kinetics of aggregation of tau and p-tau, taking advantage of the strong fluorescence emission of ThT and ThS upon binding to amyloidal protein aggregates, including tau and p-tau (Figure 2). With or without the fluorescent dye in the aggregation reaction, we observed consistent enhancement of tau aggregation by hyperphosphorylation (Figures 3&#8211;5). This stimulation is i...

Watch this Scientific Journal Video about In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein at JoVE.com

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

in-vitro-aggregation-assays-using-hyperphosphorylated-tau-protein

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18.2K Views.  Michigan State University. Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer’s disease.

Video: In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

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 ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

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 ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

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 ...