Name: fractionation for resolution of soluble and insoluble huntingtin species
Uploaded: 2018-02-27T14:30:00
Description: Watch this Scientific Journal Video about Fractionation for Resolution of Soluble and Insoluble Huntingtin Species at JoVE.com

This work was supported by the NIH (RO1-NS090390). We would also like to thank Dr. Joan Steffanfor technical assistance and discussion during the development of this assay.

Animal Ethics Statement - Experiments were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and an approved animal research protocol by the Institutional Animal Care and Use Committee (IACUC) at the University of California, Irvine, an AAALAC accredited institution. All efforts were made to minimize animal suffering.
1. Preparation of Lysis Buffers
<ol>
	<li>Prepare &#34;Soluble&#34; lysis buffer (10 mM Tris pH 7....

<ol>
	<li>Ochaba, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIAS1+Regulates+Mutant+Huntingtin+Accumulation+and+Huntington's+Disease-Associated+Phenotypes+In+Vivo.">PIAS1 Regulates Mutant Huntingtin Accumulation and Huntington's Disease-Associated Phenotypes In Vivo.</a> Neuron. 90 (3), 507-520 (2016).</li><li>La Spada, A. R., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeat+expansion+disease:+progress+and+puzzles+in+disease+pathogenesis.">Repeat expansion disease: progress and puzzles in disease pathogenesis.</a> Nat Rev Genet. 11 (4), 247-258 (2010).</li><li>Reiner, A., Dragatsis, I., Dietrich, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+neuropathology+of+Huntington's+disease.">Genetics and neuropathology of Huntington's disease.</a> Int Rev Neurobiol. 98, 325-372 (2011).</li><li>The Huntington's Disease Collaborative Research Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+gene+containing+a+trinucleotide+repeat+that+is+expanded+and+unstable+on+Huntington's+disease+chromosomes.">A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes.</a> Cell. 72 (6), 971-983 (1993).</li><li>Sassone, J., Colciago, C., Cislaghi, G., Silani, V., Ciammola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease:+the+current+state+of+research+with+peripheral+tissues.">Huntington's disease: the current state of research with peripheral tissues.</a> Exp Neurol. 219 (2), 385-397 (2009).</li><li>Weydt, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoregulatory+and+metabolic+defects+in+Huntington's+disease+transgenic+mice+implicate+PGC-1alpha+in+Huntington's+disease+neurodegeneration.">Thermoregulatory and metabolic defects in Huntington's disease transgenic mice implicate PGC-1alpha in Huntington's disease neurodegeneration.</a> Cell Metab. 4 (5), 349-362 (2006).</li><li>Schulte, J., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biological+function+of+the+Huntingtin+protein+and+its+relevance+to+Huntington's+Disease+pathology.">The biological function of the Huntingtin protein and its relevance to Huntington's Disease pathology.</a> Curr Trends Neurol. 5, 65-78 (2011).</li><li>Gipson, T. A., Neueder, A., Wexler, N. S., Bates, G. P., Housman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrantly+spliced+HTT,+a+new+player+in+Huntington's+disease+pathogenesis.">Aberrantly spliced HTT, a new player in Huntington's disease pathogenesis.</a> RNA Biol. 10 (11), 1647-1652 (2013).</li><li>Neueder, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenic+exon+1+HTT+protein+is+produced+by+incomplete+splicing+in+Huntington's+disease+patients.">The pathogenic exon 1 HTT protein is produced by incomplete splicing in Huntington's disease patients.</a> Sci Rep. 7 (1), 1307 (2017).</li><li>Arndt, J. R., Chaibva, M., Legleiter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+role+of+the+first+17+amino+acids+of+huntingtin+in+Huntington's+disease.">The emerging role of the first 17 amino acids of huntingtin in Huntington's disease.</a> Biomol Concepts. 6 (1), 33-46 (2015).</li><li>Saudou, F., Finkbeiner, S., Devys, D., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntingtin+acts+in+the+nucleus+to+induce+apoptosis+but+death+does+not+correlate+with+the+formation+of+intranuclear+inclusions.">Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions.</a> Cell. 95 (1), 55-66 (1998).</li><li>Kim, S., Kim, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+Approaches+for+Inhibition+of+Protein+Aggregation+in+Huntington's+Disease.">Therapeutic Approaches for Inhibition of Protein Aggregation in Huntington's Disease.</a> Exp Neurobiol. 23 (1), 36-44 (2014).</li><li>O'Rourke, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-2+and+PIAS1+modulate+insoluble+mutant+huntingtin+protein+accumulation.">SUMO-2 and PIAS1 modulate insoluble mutant huntingtin protein accumulation.</a> Cell Rep. 4 (2), 362-375 (2013).</li><li>Shibata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+intracellular+accumulation+of+mutant+Huntingtin+by+Beclin+1.">Regulation of intracellular accumulation of mutant Huntingtin by Beclin 1.</a> J Biol Chem. 281 (20), 14474-14485 (2006).</li><li>Apostol, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-based+assay+for+aggregation+inhibitors+as+therapeutics+of+polyglutamine-repeat+disease+and+validation+in+Drosophila.">A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila.</a> Proc Natl Acad Sci U S A. 100 (10), 5950-5955 (2003).</li><li>Wanker, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+filter+assay+for+detection+of+amyloid-like+polyglutamine-containing+protein+aggregates.">Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates.</a> Methods Enzymol. 309, 375-386 (1999).</li><li>Sontag, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Mutant+Huntingtin+Aggregation+Conformers+and+Modulation+of+SDS-Soluble+Fibrillar+Oligomers+by+Small+Molecules.">Detection of Mutant Huntingtin Aggregation Conformers and Modulation of SDS-Soluble Fibrillar Oligomers by Small Molecules.</a> J Huntingtons Dis. 1 (1), 119-132 (2012).</li><li>Grima, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+Huntingtin+Disrupts+the+Nuclear+Pore+Complex.">Mutant Huntingtin Disrupts the Nuclear Pore Complex.</a> Neuron. 94 (1), 93-107 (2017).</li><li>Sontag, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylene+blue+modulates+huntingtin+aggregation+intermediates+and+is+protective+in+Huntington's+disease+models.">Methylene blue modulates huntingtin aggregation intermediates and is protective in Huntington's disease models.</a> J Neurosci. 32 (32), 11109-11119 (2012).</li><li>Trushina, E., Rana, S., McMurray, C. T., Hua, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricyclic+pyrone+compounds+prevent+aggregation+and+reverse+cellular+phenotypes+caused+by+expression+of+mutant+huntingtin+protein+in+striatal+neurons.">Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons.</a> BMC Neurosci. 10, 73 (2009).</li><li>Shahmoradian, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRiC's+tricks+inhibit+huntingtin+aggregation.">TRiC's tricks inhibit huntingtin aggregation.</a> Elife. 2, e00710 (2013).</li><li>Kim, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+inhibition+induces+alpha-synuclein+SUMOylation+and+aggregate+formation.">Proteasome inhibition induces alpha-synuclein SUMOylation and aggregate formation.</a> J Neurol Sci. 307 (1-2), 157-161 (2011).</li><li>Goldberg, N. R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Neural+Progenitor+Transplantation+Rescues+Behavior+and+Reduces+alpha-Synuclein+in+a+Transgenic+Model+of+Dementia+with+Lewy+Bodies.">Human Neural Progenitor Transplantation Rescues Behavior and Reduces alpha-Synuclein in a Transgenic Model of Dementia with Lewy Bodies.</a> Stem Cells Transl Med. 6 (6), 1477-1490 (2017).</li></ol>

Some precautions are needed for the above protocols to ensure consistent and quantitative results. First, mHTT in both fractions will spontaneously form aggregates over time upon multiple freeze thaw cycles, particularly when in a high concentration. It is thus critical to freeze aliquots of the protein preps, and thaw only the needed volume prior to running the assay as described in the protocol above. Further, if insoluble fraction yields white precipitant upon thawing, reconstitution may be necessary by an additional ...

The disruption of protein quality control networks that ensure proper folding and degradation of cellular proteins is likely central to pathology in Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and other &#34;protein misfolding&#34; disorders2,3. A detailed understanding of the proteostasis network components and their contributions to pathology are therefore crucial to developing improved therapeutic interventions. HD is caused by the abnormal expansion of a CAG repeat within the HD gene resulting in an expanded stretch of polyglutamines (polyQ) in the huntingtin (HTT) protein4. A consistent pathological feature of HD pathogenesis is the resulting misfolding, accumulation, and aggregation of HTT in regions of the brain and in peripheral tissues, which has been shown to interfere in several ways with normal cell homeostasis and function5,6. While HTT is ubiquitously expressed, medium spiny neurons in the striatum are selectively vulnerable and most overtly affected with notable cortical atrophy also associated with HD pathogenesis.
The formation of aggregates of HTT in the brain of HD patients and animal models has typically served as a proxy marker for disease progression and dominant-negative gain of function for mHTT7. Although the precise mechanisms by which protein misfolding and aggregation may contribute to mHTT-induced toxicity remain unclear, the formation of these inclusions in the brain of HD patients and various animal models is an invariant and inevitable feature. Inclusions appear to be comprised largely of accumulated HTT fragments containing the N-terminal expanded polyQ, indicating that proteolysis and processing of full-length HTT as well as alternative splicing8,9 may play an important role in the pathogenesis of HD. N-terminal HTT fragments may constitute a pathologic form of HTT that can aggregate rapidly, nucleate, and accelerate or propagate the aggregation process10.
However, the presence of these inclusions does not necessarily correlate with HTT-induced toxicity or cell death11. HTT has been proposed to undergo an aggregation process from a soluble monomer through soluble oligomeric species and &#946;-sheet fibrils to insoluble aggregates and inclusions12. Resolving these various protein species via standard biochemical assays has been a challenge in the field due to their stability in low-detergent lysis buffer and difficulty in visualizing using standard biochemical assays. Thus, methodological considerations are critical in detecting the degree and pattern of accumulated and aggregated mHTT.
The protocol presented here provides a method to visualize several intermediates of HTT, specifically the formation of an insoluble HMW HTT species that appears to closely track with HD pathogenesis and disease progression1,13,14. Being able to resolve and track multiple species of mHTT provides researchers a biochemical tool to study disease pathogenesis and evaluate potential therapeutic interventions through their modulation and impact on disease pathogenesis.

<table>
	<tbody>
		<tr>
			<td>Sterile Filter</td>
			<td>Millipore</td>
			<td>SCGP505RE</td>
			<td>Screw cap, sterile vaccum filter</td>
		</tr>
		<tr>
			<td>1 mL Tissue Grinder, Dounce</td>
			<td>Wheaton</td>
			<td>357538</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Qsonica</td>
			<td>Model Q125</td>
			<td></td>
		</tr>
		<tr>
			<td>DC Protein Assay</td>
			<td>Biorad</td>
			<td>5000111</td>
			<td>Comparable to Lowry assay</td>
		</tr>
		<tr>
			<td>Tris 1M buffer solution</td>
			<td>Alfa Aesar</td>
			<td>J60636</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 5M solution</td>
			<td>Teknova</td>
			<td>S0251</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>20% SDS solution</td>
			<td>Teknova</td>
			<td>S0295</td>
			<td></td>
		</tr>
		<tr>
			<td>N-ethylmaleimide</td>
			<td>Sigma</td>
			<td>E1271</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethylsulfony flouride</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>create 100mM stock solution in 100%EtOH, store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td>Create 0.5M stock solution in water</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td>Create 10mg/ml stock solution in water</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
			<td>Create 10mg/ml stock solution in water</td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>Sigma</td>
			<td>S4504</td>
			<td>Create 500mM stock solution in water</td>
		</tr>
		<tr>
			<td>Anti-HTT</td>
			<td>Millipore</td>
			<td>MAB5492</td>
			<td>Use 1:1000 for western blot, 1:500 for filter retardation assay</td>
		</tr>
		<tr>
			<td>Anti-GAPDH</td>
			<td>Novus Biologicals</td>
			<td>NB100-56875</td>
			<td>Use 1:1000 for soluble western blot</td>
		</tr>
	</tbody>
</table>

fractionation for resolution of soluble and insoluble huntingtin species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

Resolution of soluble and insoluble cell lysates following fractionation can be detected using western analysis and filter retardation assays (Figure 2). As an example, HEK293T cells were transfected using transfection reagent (e.g., lipofectamine 2000), with HTT exon 1 encoding cDNA containing 97 glutamine repeats15 followed by the poly proline rich region, and these cells were allowed to express for 44 h. Cells were treated ...

Watch this Scientific Journal Video about Fractionation for Resolution of Soluble and Insoluble Huntingtin Species at JoVE.com

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

The overall goal of this procedure is to describe a useful tool for visualization, characterization, and quantification of huntingtin protein flux for the analysis of protein homeostasis in Huntington's disease model systems. This method can help us understand questions in protein homeostasis and neurodegenerative disease fields such as understanding the impact of certain therapeutic interventions on disease pathogenesis. The main advantage of this technique is that it allows investigators to isolate and resolve insoluble protein species for quantitative analysis.Though this method can provide insight into biochemical changes during Huntington's disease pathogenesis, it can also be applied to other protein misfolding diseases such as Parkinson's and Alzheimer's disease. To begin this procedure, label two sets of tubes for each sample as soluble and insoluble. Prepare a lysis buffer immediately prior to sample lysis in the soluble tube by adding the appropriate amount of inhibitors.Next, add ice cold working soluble lysis buffer supplemented with protease inhibitors to the tissue. For mouse brain tissue, slowly dounce 30 times in a one milliliter glass douncer. Be careful not to break the surface of liquid as bubbles will form and may yield incomplete homogenization.Then pipette it into the insoluble-labeled tube on ice. For cell lines, cells were plated at five times 10 to the fifth cells in each six-well plate and grown to near confluency. Afterward, rinse the cells once with cold 1X PBS and remove it.Then apply minimal volume of lysis buffer to the cells and lift them with a cell scraper. Next, transfer homogenate or cell lysate into the labeled insoluble tube and lyse on ice for one hour. For cell lysates, triturate several times to break up cell clumps prior to starting incubation and avoid forming bubbles.Briefly pulse vortex each sample for one second halfway through lysing. After that, centrifuge the samples at four degrees Celsius at 15, 000 x g for 20 minutes. Then remove the supernatant.Be careful not to disturb the pellet or cloudy layer as this will contaminate the fraction. Pipette the supernatant as soluble fraction into the soluble-labeled tube and keep it on ice. Keeping track of the soluble and insoluble lysis buffers is key.Switching the buffers or placing the insoluble buffer or samples on ice will be detrimental to the outcome of the protocol. Wash the pellet with 500 microliters of lysis buffer and centrifuge at four degrees Celsius at 15, 000 x g for five minutes. Repeat the wash one more time.Subsequently, remove all the remaining wash buffer leaving only the tissue pellet. Next, resuspend the pellet with lysis buffer supplemented with 4%SDS. Sonicate each sample for 30 seconds at room temperature with a probe sonicator.Sonicating small volumes for a long period of time is technically challenging. Be careful not to lose any sample by emulsifying or spraying while completing this step. Afterward, boil the samples for 30 minutes.Then centrifuge them briefly at 6, 000 x g. Subsequently, perform detergent compatible protein assay or Lowry protein assay to measure protein concentration. Following that, aliquot the samples into 50 microliter batches to avoid freeze/thaw issues.In this step, perform western blots by diluting 30 micrograms of sample at a ratio of one to one in loading buffer. Boil the samples for five minutes and centrifuge them briefly at 6, 000 x g. Next, analyze the fractions by SDS page and western blot.Stain the membrane with whole protein stain to visualize protein loading efficiency. Here are the soluble and insoluble fractions from HEK 293T cell lysates resolved on a 4-12%Bis-Tris and 3-8%Tris-Acetate page gels respectively. Soluble fraction normalized to GAPDH loading control shows fluctuation of mutant huntingtin exon 1 soluble monomer based on exposure to protease inhibitor MG132.Insoluble fraction shows that high molecular weight accumulated species of mutant huntingtin is also modulated by MG132 treatment. And here are the samples of soluble and insoluble fractions from HeLA cell lysates using western blot and filter retardation analysis. Data shows that the presence of an over expressed SUMO isoform increases both high molecular weight mutant and fibrillary insoluble mutant huntingtin in the insoluble fraction.This figure shows the regulation of high molecular weight mutant huntingtin accumulation after modulating a potential therapeutic in the insoluble fractionation from HeLa cells over expressing 97Q huntingtin exon 1. Therapeutic target PIAS1 was either silenced with an siRNA towards PIAS1 or over expressed. Insoluble fractions from mouse striata treated with microRNA against PIAS1 and resolved on 3-8%Tris-Acetate page gels show modulation of high molecular weight insoluble huntingtin.Don't forget that working with NEM can be extremely hazardous and taking precautions such as wearing a face mask should always be taken while performing this protocol. Once mastered, this technique from lysate to end of fractionation can be done in about three to four hours depending on the number of samples. An additional hour should be allowed for protein quantification and aliquoting.Following this procedure, other methods such as agarose gel electrophoresis or filter retardation can be performed in order to detect soluble oligomers or insoluble fibrils respectively. After watching this video, you should have a good understanding of how to isolate and resolve insoluble misfolded proteins for quantitative analysis.

fractionation-for-resolution-soluble-insoluble-huntingtin

Research

JoVE Journal

Biochemistry

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS3) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch.
The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (&#8805;42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of the strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

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

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

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

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

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...