Name: sds-page/immunoblot detection of a&beta; multimers in human cortical tissue homogenates using antigen-epitope retrieval
Uploaded: 2010-04-23T04:53:00
Description: Watch this Scientific Journal Video about SDS-PAGE/Immunoblot Detection of AÎ² Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval at JoVE.com

Many thanks to Elaine Pranski and Carolyn Suwyn for excellent technical assistance and to Harry LeVine III, M. Paul Murphy, and Marla Gearing for important conversations. Funding was provided by RR-00165, PO1AG026423, P50AG025688, AG030539, the Woodruff Foundation and the Emory University Research Committee.

Part 1: Preparation of clarified tissue homogenates
<ol>
 <li>Dounce homogenize unfixed cortical tissue in 4 times volume of ice-cold buffer (0.1M phosphate-buffered saline [Ca++- and Mg++-free] plus 2x protease inhibitor cocktail [Santa Cruz]) with approximately 30 even pestle strokes (i.e. add 400&mu;l buffer to 100mg tissue). </li>
 <li>Spin homogenates for 5 minutes at 3,000g (4&deg;C). Carefully remove clarified supernatants and store aliquots of these extracts at -80&deg;C until use. </li>
 <li>Determin...

<ol>
	<li>Hardy, J., 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=The+amyloid+hypothesis+of+Alzheimer's+disease:+progress+and+problems+on+the+road+to+therapeutics.">The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.</a> Science. 297, 353-356 (2002).</li><li>Haass, C., 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=Soluble+protein+oligomers+in+neurodegeneration:+lessons+from+the+Alzheimer's+amyloid+beta-peptide.">Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.</a> Nat. Rev. Mol. Cell. Biol. 8, 101-112 (2007).</li><li>Walker, L. C., LeVine, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cerebral+proteopathies.">The cerebral proteopathies.</a> Neurobiol. Aging. 21, 559-561 (2000).</li><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=Aging,+amyloid,+and+Alzheimer's+disease:+a+perspective+in+honor.">Aging, amyloid, and Alzheimer's disease: a perspective in honor.</a> of Carl Cotman. Neurochem. Res. 28, 1705-1713 (2003).</li><li>Walsh, D. M., 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=Abeta+oligomers+-+a+decade+of+discovery.">Abeta oligomers - a decade of discovery.</a> J. Neurochem. 101, 1172-1184 (2007).</li><li>Swerdlow, P. S., Finley, D., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+immunoblot+sensitivity+by+heating+of+hydrated+filters.">Enhancement of immunoblot sensitivity by heating of hydrated filters.</a> Anal. Biochem. 156, 147-153 (1986).</li><li>Ida, N., Hartmann, T., Pantel, J., Schr&#246;der, J., Zerfass, R., F&#246;rstl, H., Sandbrink, R., Masters, C. L., Beyreuther, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+hetergeneous+A4+peptides+in+human+cerebrospinal+fuid+and+blood+by+a+newly+developed+sensitive+Western+blot+assay.">Analysis of hetergeneous A4 peptides in human cerebrospinal fuid and blood by a newly developed sensitive Western blot assay.</a> J. Biol. Chem. 271, 22908-22914 (1996).</li></ol>

Despite the importance of A&beta; aggregation in the pathogenesis of Alzheimer's disease1,4,5 , few studies have described or quantified the distribution of diverse A&beta; multimers in human cortical tissue samples2. Commonly used immunohistochemical techniques do not allow for the discrimination of distinct multimeric A&beta; species in fixed cortical tissue. In unfixed cortical tissue homogenates, A&beta; multimers can be separated and biochemically assessed using gel electrophoresis and antibod...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Complete Protease Inhibitor Cocktail Tablets</td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-29130</td>
<td>1 tablet in 25ml buffer</td>
</tr>
<tr>
<td>BCA Protein Assay kit</td>
<td>&nbsp;</td>
<td>ThermoFisher Scientific</td>
<td>23225</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell and XCell II Blot Module Kit CE Mark</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EI0002</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Sample Buffer (2X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>NuPAGE Sample Reducing Agent (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>NP0004</td>
<td>&nbsp;</td>
<tr>
<td>SeeBlue Plus2 Pre-Stained Standard</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC5925</td>
<td>&nbsp;</td>
<tr>
<td>Novex 10-20% Tricine Gel 1.0 mm, 10 well</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EC6625BOX</td>
<td>&nbsp;</td>
<tr>
<td>Nitrocellulose membranes, 0.2 &mu;m pore size</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2000</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tris-Glycine Transfer Buffer (25X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC3675</td>
<td>&nbsp;</td>
<tr>
<td>SimplyBlue SafeStain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6060</td>
<td>Will not interfere with immunostaining</td>
</tr>
<tr>
<td>ATX Ponceau S Red staining solution</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>09276</td>
<td>Will not interfere with immunostaining</td>
</tr>
<tr>
<td>Kapak heat sealable plastic sample pouches</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>0181225AA</td>
<td>&nbsp;</td>
<tr>
<td>6E10 mouse monoclonal antibody to A&beta;(1-16)</td>
<td>&nbsp;</td>
<td>Covance</td>
<td>SIG-39320</td>
<td>Dilute 1:1,000 up to 1:5,000 for WB</td>
</tr>
<tr>
<td>Tween 20</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P2287</td>
<td>&nbsp;</td>
<tr>
<td>ECL Mouse IgG, HRP-Linked Whole A&beta; (from sheep)</td>
<td>&nbsp;</td>
<td>GE Healthcare Life Sciences</td>
<td>NA931-1ML</td>
<td>Dilute at 1:10,000</td>
</tr>
<tr>
<td>SuperSignal West Pico Chemiluminescent Substrate</td>
<td>&nbsp;</td>
<td>ThermoFisher</td>
<td>34077</td>
<td>&nbsp;</td>
<tr>
<td>Kodak Biomax MR Film</td>
<td>&nbsp;</td>
<td>Carestream Health</td>
<td>870 1302</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

sds-page/immunoblot detection of a&beta; multimers in human cortical tissue homogenates using antigen-epitope retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. A&beta; is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into &beta;-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble A&beta; multimers are more toxic than fibrillar A&beta; amyloid2. The identification and quantification of low- and high-molecular weight multimeric A&beta; species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring A&beta; multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with A&beta;-specific antibodies. However, the separation and detection of multimeric A&beta; requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the A&beta; peptide. Using this protocol, we consistently detect A&beta; monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

Watch this Scientific Journal Video about SDS-PAGE/Immunoblot Detection of AÎ² Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval at JoVE.com

SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the A&beta; peptide. Using this protocol, we consistently detect monomeric and multimeric A&beta; in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

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