This work is supported by grants to W.C. from National Institutes of Health Grant AI074809 and The University of Texas M. D. Anderson Cancer Center Institutional Research Grant Program.

1. Prepare MES Buffer
<ol>
	<li>Use MES buffer (0.1 M 2-(N-morpholino)ethanesulfonic acid and 0.9 M NaCl) to prepare both protein-only amyloid as well as stabilized soluble protein oligomers. Weigh 1.95 g of MES and 5.27 g of NaCl. Add H2O and adjust pH to 4.7. Make up the final volume to 100 ml. Store at 4&#186; C for up to 6 months.</li>
</ol>
2. Prepare Protein-only Amyloid Directly from Native Proteins
<ol>
	<li>Weigh protein of choice (any native protein or peptide) and reconstitute to 10 mg/ml in MES buffer.</li>
	<li>Incubate at 65 &#186;C in a waterbath for 4 hr. By the end of the period, white precipitate is visible in solution.</li>
	<li>Neutralize the sample with 10% (v/v) of Tris-HCl (1 M, pH 10.5).</li>
	<li>Dialyze the protein in a dialysis cassette overnight to exchange to the desired buffer for further analysis.</li>
	<li>A control sample of native protein can be prepared by following steps 2.1, 2.3, and 2.4 without heat precipitation (step 2.2).</li>
</ol>
3. Prepare Hybrid Amyloid from Stabilized Soluble Oligomers of Native Proteins
<ol>
	<li>Prepare stabilized soluble protein oligomers
	<ol>
		<li>Weigh protein of choice and reconstitute to 10 mg/ml in MES buffer. Leave at room temperature for 15 min.</li>
		<li>Weigh 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and prepare a fresh stock of 20 mg/ml in MES buffer.</li>
		<li>Mix protein and EDC solutions at a ratio of 2:5 (v/v), i.e. 2 mg of protein for every 10 mg of EDC.</li>
		<li>Incubate at room temperature for 2 hr.</li>
		<li>Neutralize the sample with 10% (v/v) of Tris-HCl (1 M, pH 10.5).</li>
		<li>Dialyze the protein in a dialysis cassette overnight to exchange to the desired buffer for further analysis.</li>
		<li>Store the stabilized soluble protein oligomer at 4 &#186;C up to 4 weeks.</li>
	</ol>
	</li>
	<li>Prepare hybrid amyloids from stabilized soluble protein oligomer.
	<ol>
		<li>To prepare DNA-containing amyloid, mix DNA and stabilized soluble protein oligomer at 1:1 ratio (w/w). Leave at 4 &#186;C for at least 2 hr. During this period, insoluble precipitate is visible in solution when the initial protein concentration is 0.5 mg/ml or higher.</li>
		<li>To prepare RNA-containing amyloid, mix RNA and stabilized soluble protein oligomer at 1:1 ratio (w/w). Leave at 4 &#186;C for at least 2 hr. During this period, insoluble precipitate is visible in solution when the initial protein concentration is 0.5 mg/ml or higher.</li>
		<li>To prepare heparin-containing amyloid, mix heparin and stabilized soluble protein oligomer at 1:1 ratio (w/w). Leave at 4 &#186;C for at least 2 hrs. During this period, insoluble precipitate is visible in solution when the initial protein concentration is 0.5 mg/ml or higher.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+proteins+in+fatal+ways.">Folding proteins in fatal ways.</a> Nature. 426, 900-904 (2003).</li><li>Schnabel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+folding:+The+dark+side+of+proteins.">Protein folding: The dark side of proteins.</a> Nature. 464, 828-829 (2010).</li><li>Goldberg, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+degradation+and+protection+against+misfolded+or+damaged+proteins.">Protein degradation and protection against misfolded or damaged proteins.</a> Nature. 426, 895-899 (2003).</li><li>Chiti, F., Dobson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding,+functional+amyloid,+and+human+disease.">Protein misfolding, functional amyloid, and human disease.</a> Annu. Rev. Biochem. 75, 333-366 (2006).</li><li>Goldschmidt, L., Teng, P. K., Riek, R., Eisenberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+the+amylome,+proteins+capable+of+forming+amyloid-like+fibrils.">Identifying the amylome, proteins capable of forming amyloid-like fibrils.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 3487-3492 (2010).</li><li>Fowler, D. M., Koulov, A. V., Balch, W. E., Kelly, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+amyloid+-+from+bacteria+to+humans.">Functional amyloid - from bacteria to humans.</a> Trends Biochem. Sci. 32, 217-224 (2007).</li><li>Chapman, M. R., 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=Role+of+Escherichia+coli+Curli+Operons+in+Directing+Amyloid+Fiber+Formation.">Role of Escherichia coli Curli Operons in Directing Amyloid Fiber Formation.</a> Science. 295, 851-855 (2002).</li><li>Barnhart, M. M., Chapman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curli+Biogenesis+and+Function.">Curli Biogenesis and Function.</a> Annu. Rev. Microbiol. 60, 131-147 (2006).</li><li>Claessen, D., 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+n	olved+in+aerial+hyphae+formation+in+Streptomyces+coelicolor+by+forming+amyloid-like+fibrils.">A n	olved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils.</a> Genes Dev. 17, 1714-1726 (2003).</li><li>Maji, S. K., 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=Functional+Amyloids+As+Natural+Storage+of+Peptide+Hormones+in+Pituitary+Secretory+Granules.">Functional Amyloids As Natural Storage of Peptide Hormones in Pituitary Secretory Granules.</a> Science. 325, 328-332 (2009).</li><li>Badtke, M. P., Hammer, N. D., Chapman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Amyloids+Signal+Their+Arrival.">Functional Amyloids Signal Their Arrival.</a> Sci. Signal. 2, pe43 (2009).</li><li>Li, 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=The+RIP1/RIP3+Necrosome+Forms+a+Functional+Amyloid+Signaling+Complex+Required+for+Programmed+Necrosis.">The RIP1/RIP3 Necrosome Forms a Functional Amyloid Signaling Complex Required for Programmed Necrosis.</a> Cell. 150, 339-350 (2012).</li><li>Hou, F., 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=MAVS+forms+functional+prion-like+aggregates+to+activate+and+propagate+antiviral+innate+immune+response.">MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.</a> Cell. 146, 448-461 (2011).</li><li>Stefani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+biophysical+features+of+both+oligomer/fibril+and+cell+membrane+in+amyloid+cytotoxicity.">Biochemical and biophysical features of both oligomer/fibril and cell membrane in amyloid cytotoxicity.</a> FEBS J. 277, 4602-4613 (2010).</li><li>Glabe, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+classification+of+toxic+amyloid+oligomers.">Structural classification of toxic amyloid oligomers.</a> J. Biol. Chem. 283, 29639-29643 (1074).</li><li>Di Domizio, 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=Binding+with+nucleic+acids+or+glycosaminoglycans+converts+soluble+protein+oligomers+to+amyloid.">Binding with nucleic acids or glycosaminoglycans converts soluble protein oligomers to amyloid.</a> J. Biol. Chem. 287, 736-747 (2012).</li><li>Zhang, X., Li, J. -. P., Lijuan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+proteoglycans+in+amyloidosis.">Heparan sulfate proteoglycans in amyloidosis.</a> Prog. Mol. Biol. Transl. Sci. 93, 309-334 (2010).</li><li>Jim&#233;nez, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-DNA+Interaction+at+the+Origin+of+Neurological+Diseases:+A+Hypothesis.">Protein-DNA Interaction at the Origin of Neurological Diseases: A Hypothesis.</a> J. Alzheimer's Dis. 22, 375-391 (2010).</li><li>Bhattacharya, M., Jain, N., Mukhopadhyay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+Mechanism+of+Aggregation+and+Fibril+Formation+from+Bovine+Serum+Albumin.">Insights into the Mechanism of Aggregation and Fibril Formation from Bovine Serum Albumin.</a> J. Phys. Chem. B. 115, 4195-4205 (2011).</li><li>Vetri, V., 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=Bovine+Serum+Albumin+protofibril-like+aggregates+formation:+Solo+but+not+simple+mechanism.">Bovine Serum Albumin protofibril-like aggregates formation: Solo but not simple mechanism.</a> Arch. Biochem. Biophysics. 508, 13-24 (2011).</li><li>Bucciantini, 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=Inherent+toxicity+of+aggregates+implies+a+common+mechanism+for+protein+misfolding+diseases.">Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.</a> Nature. 416, 507-511 (2002).</li><li>McParland, V. J., Kalverda, A. P., Homans, S. W., Radford, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+properties+of+an+amyloid+precursor+of+b2-microglobulin.">Structural properties of an amyloid precursor of b2-microglobulin.</a> Nat. Struct. Mol. Biol. 9, 326-331 (2002).</li><li>Chiti, F., 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=Designing+conditions+for+in+vitro+formation+of+amyloid+protofilaments+and+fibrils.">Designing conditions for in vitro formation of amyloid protofilaments and fibrils.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 3590-3594 (1999).</li><li>McParland, V. 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=Partially+Unfolded+States+of+b2-Microglobulin+and+Amyloid+Formation+in+vitro.">Partially Unfolded States of b2-Microglobulin and Amyloid Formation in vitro.</a> Biochemistry. 39, 8735-8746 (2000).</li><li>Liu, K., Cho, H. S., Lashuel, H. A., Kelly, J. W., Wemmer, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+glimpse+of+a+possible+amyloidogenic+intermediate+of+transthyretin.">A glimpse of a possible amyloidogenic intermediate of transthyretin.</a> Nat. Struct. Mol. Biol. 7, 754-757 (2000).</li><li>Pfefferkorn, C. M., McGlinchey, R. P., Lee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+pH+on+aggregation+kinetics+of+the+repeat+domain+of+a+functional+amyloid.">Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid.</a> Pmel17. Proc. Natl. Acad. Sci. U.S.A. 107, 21447-21452 (2010).</li><li>Di Domizio, 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=Nucleic+acid-containing+amyloid+fibrils+potently+induce+type+I+interferon+and+stimulate+systemic+autoimmunity.">Nucleic acid-containing amyloid fibrils potently induce type I interferon and stimulate systemic autoimmunity.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 14550-14555 (2012).</li><li>Halle, A., Hornung, V., Petzold, G. C., Stewart, C. R., Monks, B. G., Reinheckel, T., Fitzgerald, K. A., Latz, E., Moore, K. J., Golenbock, D. 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+NALP3+inflammasome+is+involved+in+the+innate+immune+response+to+amyloid-&#946;.">The NALP3 inflammasome is involved in the innate immune response to amyloid-&#946;.</a> Nat. Immunol. 9, 857 (2008).</li><li>Masters, S. L., O'Neill, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-associated+amyloid+and+misfolded+protein+aggregates+activate+the+inflammasome.">Disease-associated amyloid and misfolded protein aggregates activate the inflammasome.</a> Trends Mol. Med. 17, 276-282 (2011).</li></ol>

We have nothing to disclose.

The method described here offers a rapid and flexible means to prepare amyloid fibrils in vitro from virtually any protein or peptide of choice. Several different types of amyloid can be reliably prepared - protein-only fibrils or hybrid aggregates containing DNA, RNA, &#160;or glycosaminoglycans. The procedures involved are simple, straight forward, and do not require advanced technical training. The amyloid prepared by this method is quite stable and can be safely stored at 4 &#186;C or frozen at -80 &#186;C f...

Proteins are the most abundant biological macromolecules present in all types of cells. They occur in a great variety of sizes, structures, and post-translational modifications, and fulfill an enormous range of important biological functions when in their native forms. More than two dozens of aberrant polypeptides have been implicated in numerous human pathological conditions, such as Alzheimer&#39;s disease, Parkinson disease, &#160;and Type 2 Diabetes1-4. The terminal misfolded proteins accumulate as amyloid fibrils&#160;the insoluble stable aggregates that occur extracellularly or intracellularly.
Despite the implication of specific proteins in certain diseases, increasing evidence supports the notion that all polypeptides have intrinsic properties that enable amyloid transformation. A genome-wide sequence survey identified the &#8220;amylome&#8221;, by which amyloid-prone fragments constitute roughly 15% of all coding peptide segments from E. coli to humans5. Accordingly, an increasing number of functional amyloids, which participate in various important cellular processes, have been discovered in recent years. For example, bacteria assemble amyloids to form biofilm and spore structures that are critical for their survival and pathogenesis6-9. Peptide hormones form amyloid deposits during storage within mammalian secretory granules before being released, further implying that the protein amyloid form can serve beneficial biological functions6,10,11. Moreover, proteins can also assemble into amyloid fibrils to relay critical cellular signals12,13.
For many years, the most studied amyloids are prepared from individual peptides that are associated with human diseases, such as amyloid beta or prion peptide. These peptides usually lack well defined structure and spontaneously form amyloid in solution over time, a process mediated by a partially misfolded intermediate that serves as the precursor of insoluble amyloid. The precursor of amyloid is also called soluble protein oligomer, which is rare and unstable when generated from natural peptides14,15. However, as described earlier, almost any protein in principle can adopt amyloid conformation under the appropriate conditions. We recently established a method to prepare stabilized soluble oligomers of native proteins, which readily form amyloid in the presence of various cofactors16. The resulting amyloid fibrils reflect the complex nature of the terminal protein misfolding aggregates17,18. Here we use the term protein-only amyloid to refer the protein fibrils without cofactors and hybrid amyloid for the fibrils containing nonproteinaceous components.
Previously, various methods have been developed to generate amyloid in vitro by applying conditions known to favor protein misfolding, such as high temperature, hydrophobic environment, and pH variations19-23. However, they associate with various problems, such as low efficiency, reversible folding, large batch variations or slow kinetics. The approach described here is reproducible, easy to scale up, and allows one to precisely control the timing and condition of amyloid conversion.

<table border="0" cellpadding="0" cellspacing="0" width="795">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">2-(N-morpholino)ethanesulfonic acid (MES)</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:164px;">M8250</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">NaCl</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:164px;">BP358-10</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">1-ethyl-3-[3-dimethyl-aminopropyl] carbodiimide hydrochloride (EDC)</td>
			<td style="width:145px;">Thermo/Pierce</td>
			<td style="width:164px;">22980</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">DNA from salmon sperm</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:164px;">D1626</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">RNA from torula yeast</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:164px;">R6625</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">Heparin from porcine intestinal mucosa</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:164px;">H3149</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">Tris base</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:164px;">BP152-1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">Equipment:</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:487px;">Material Name</td>
			<td style="width:145px;">Company</td>
			<td style="width:164px;">Catalogue Number</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:487px;">Water bath</td>
			<td style="width:145px;">Fisher Science</td>
			<td style="width:164px;">Isotemp 210</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:487px;">Slide-A-Lyzer dialysis cassette</td>
			<td style="width:145px;">Thermo/Pierce</td>
			<td style="width:164px;">66380</td>
		</tr>
	</tbody>
</table>

rapid generation of amyloid from native proteins in vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

The ability to convert native proteins directly to amyloid relies on the conformational change incurred at high temperature in the MES buffer. Precipitation in the solution is a good indication of protein aggregation and possibly amyloid formation. On the other hand, to allow soluble protein oligomer to stabilize, EDC is used to crosslink the proteins to solidify the oligomeric conformation induced in MES buffer. As a result, the stabilized protein oligomers are multimeric proteins (Figure 1), distinct f...

Watch this Scientific Journal Video about Rapid Generation of Amyloid from Native Proteins In vitro at JoVE.com

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

rapid-generation-of-amyloid-from-native-proteins-in-vitro

Research

JoVE Journal

Biology

6.1K visualizações.  The University of Texas MD Anderson Cancer Center. Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Vídeo: Rapid Generation of Amyloid from Native Proteins In vitro

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Eletroforese em Gel de Poliacrilamida azul Nativo (BN-PAGE) para Análise de Complexos Multiproteicos de Lisados ​​Celular

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Biologia

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Geração rápida e eficiente de neurônios a partir de células-tronco pluripotentes humanas, em um formato de placa Multititre

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Análise Genética rápida de epitelial-mesenquimal sinalização durante a regeneração do cabelo

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Rápida e eficiente Zebrafish Genotipagem Usando PCR com alta resolução Análise Melt

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Geração de Myospheres De hESCs por epigenética Reprogramação

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente Geração induzida pelo homem células-tronco pluripotentes a partir de células somáticas humanas com Sendai-vírus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

In Vitro Pâncreas Organogenesis de dispersos rato embrionárias Progenitores

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Detecção de proteínas de ligação a ARN pela<em&gt; In Vitro</em&gt; RNA Pull-down em Cultura adipócito

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Isolamento de filtração de Ácidos Nucleicos: Um Método de extracção de DNA simples e rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Análise de microbiota usando PCR de duas etapas e sequenciamento do gene 16S rRNA de próxima geração

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...