Bitan:Extraction of mice brains for α-syn ELISA

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<p class=MsoNormal>Brain Extraction Protocol</p>

<p class=ListParagraph style='text-indent:-.25in'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>Make lysis buffer; keep on ice</p>

<p class=ListParagraph style='margin-left:1.0in;text-indent:-.25in'><span style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span>1 tablet Com antiphos into 1 mL lyis buffer = 25X stock 40ul/1mL</p>

<p class=ListParagraph style='margin-left:1.0in;text-indent:-.25in'><span style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;

</span></span>10mL lysis buffer + 1 phostop tablet + 40uL Com</p>

<p class=ListParagraph style='margin-left:1.0in;text-indent:-.25in'><span style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span>10mL lysis + 1% triton</p>

<p class=ListParagraph style='text-indent:-.25in'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>Get brains, keep on ice, weigh scale in B117B</p>

<p class=ListParagraph style='text-indent:-.25in'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>Put brains in 5mL glass tube</p>

<p class=ListParagraph style='text-indent:-.25in'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>1mL lysis + phsph + Com into tube</p>

<p class=ListParagraph style='text-indent:-.25in'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>Get beakers of ddH2O and 100% EtOH</p>

<p class=ListParagraph style='margin-left:1.0in;text-indent:-.25in'><span style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span>Use pellet/pestle motor to crush</p>

<p class=ListParagraph style='margin-left:1.0in;text-indent:-.25in'><span style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;

</span></span>Clean tip in water at EtOH and wipe between brains</p>

<p class=ListParagraph style='text-indent:-.25in'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>Sonicate on low (~1 or 2) till looks homogenous</p>

<p class=ListParagraph style='margin-left:1.0in;text-indent:-.25in'><span style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span>keep on ice while sonicating</p>

<p class=ListParagraph style='margin-left:1.0in;text-indent:-.25in'><span style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span>sonicate in bursts otherwise it heats solution and can denature protein</p>

<p class=ListParagraph style='margin-left:1.0in;text-indent:-.25in'><span style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span>transfer to 1.5mL tube (might need dropper for chunks)</p>

<p class=ListParagraph style='margin-left:1.5in;text-indent:-.25in'><span style='font-family:Wingdings'>§<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span>might want to wait 10-15 min before spinning</p>

<p class=ListParagraph style='text-indent:-.25in'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>Spin </p>

<p class=ListParagraph style='text-indent:-.25in'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;

</span>Collect</p>

<p class=ListParagraph style='text-indent:-.25in'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>Freeze all samples</p>

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