Mar 20, 2024

Public workspaceGENERATION OF LYSATE INOCULATION MATERIALS

DOI
dx.doi.org/10.17504/protocols.io.4r3l22d2ql1y/v1
  • Scott Vermilyea1
  • 1University of Minnesota
Jane Balster
Open access
DOI: dx.doi.org/10.17504/protocols.io.4r3l22d2ql1y/v1
Protocol Citation: Scott Vermilyea 2024. GENERATION OF LYSATE INOCULATION MATERIALS. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22d2ql1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 21, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95767
Keywords: ASAPCRN
Abstract
This protocol details inoculation lysate preparation.
Attachments
1035 - 2660.docx
1035 - 2660.docx
18KB
Materials

Lysis buffer:

AB
Sucrose250 mM
HEPES20mM
KCl10mM
MgCl21.5mM
EDTA2 mM
Protease-inhibitor cocktail


Soluble/Insoluble lysate preparation from harvested brainstem/spinal cord tissue
Soluble/Insoluble lysate preparation from harvested brainstem/spinal cord tissue
Acquire the tissue from 4-month-old asymptomatic TgA53T (Line G2-3) and end-stage tissue harvest and store at Temperature-80 °C prior to use.

Weigh the tissue and suspend it in 0.9% sterile saline (1:10 w/vol).
Homogenize and centrifuge for Duration00:05:00 at Centrifigation3000 x g at Temperature4 °C . The supernatant (S3000) is obtained.



5m
Centrifigation
Centrifuge S3000 for Duration00:45:00 at Centrifigation150000 x g at Temperature4 °C . The supernatant (S150) – Highly soluble fraction is obtained.

45m
Centrifigation
Wash pellet and resuspend in sterile saline (half of original volume) by sonication (3 x 10 min pulses). Resuspended pellet (P150) – Insoluble fraction is obtained.


Wash
Wash pellet and resuspend in sterile saline (half of original volume) by sonication for Duration00:00:10 pulses) (1/3).
10s
Wash pellet and resuspend in sterile saline (half of original volume) by sonication for Duration00:00:10 pulses) (2/3).
10s
Wash pellet and resuspend in sterile saline (half of original volume) by sonication for Duration00:00:10 pulses) (3/3).
10s
Endoplasmic Reticulum (ER)-enriched fractionation
Endoplasmic Reticulum (ER)-enriched fractionation
Homogenize freshly harvested tissues (1:10 w/vol) in lysis buffer (refer material section).
Centrifuge at Centrifigation1000 x g and collect the supernatant.

Centrifigation
Centrifuge at Centrifigation10000 x g . The pellet containing mitochondria is obtained.

Centrifigation
Centrifuge supernatant at Centrifigation100000 x g . The supernatant containing cytosol and pellet containing ER are obtained.

Centrifigation