Bitan:Extraction of mice brains for α-syn ELISA

       Brain Extraction Protocol



Brain Extraction Protocol

- Make lysis buffer; keep on ice

o 1 tablet Com antiphos into 1 mL lyis buffer = 25X stock 40ul/1mL

o

10mL lysis buffer + 1 phostop tablet + 40uL Com

o 10mL lysis + 1% triton

<p class=ListParagraph style='text-indent:-.25in'>- Get brains, keep on ice, weigh scale in B117B

<p class=ListParagraph style='text-indent:-.25in'>- Put brains in 5mL glass tube

<p class=ListParagraph style='text-indent:-.25in'>- 1mL lysis + phsph + Com into tube

<p class=ListParagraph style='text-indent:-.25in'>- Get beakers of ddH2O and 100% EtOH

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

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

Clean tip in water at EtOH and wipe between brains

<p class=ListParagraph style='text-indent:-.25in'>- Sonicate on low (~1 or 2) till looks homogenous

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

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

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

<p class=ListParagraph style='margin-left:1.5in;text-indent:-.25in'><span style='font-family:Wingdings'>§ might want to wait 10-15 min before spinning

<p class=ListParagraph style='text-indent:-.25in'>- Spin

<p class=ListParagraph style='text-indent:-.25in'>-

Collect

<p class=ListParagraph style='text-indent:-.25in'>- Freeze all samples