Mar 20, 2024

Public workspaceProtein Lysate Preparation Protocol (Tissue)

DOI
dx.doi.org/10.17504/protocols.io.n2bvj3k3xlk5/v1
  • Scott Vermilyea1
  • 1University of Minnesota
Jane Balster
Open access
DOI: dx.doi.org/10.17504/protocols.io.n2bvj3k3xlk5/v1
Protocol Citation: Scott Vermilyea 2024. Protein Lysate Preparation Protocol (Tissue). protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj3k3xlk5/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 26, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95763
Keywords: ASAPCRN
Abstract
This protocol details the preparation of protein lysate of the tissue sample.

Attachments
Western Blot and Lysate Prep Protocol.docx
Western Blot and Lys...
19KB
Protein Lysate Preparation (Tissue)
Protein Lysate Preparation (Tissue)
4h 20m
If tissue stored at Temperature-80 °C , place in Temperature-20 °C for at least Duration04:00:00 or DurationOvernight prior to homogenization.

4h
Overnight
Weigh tissue out in mg.
Add in 10 volumes of 1X TNE (e.g. Amount10 µL of TNE per Amount1 mg of tissue).
Either by mechanical (Dounce) or homogenizer machine, homogenize tissue gently and TemperatureOn ice .

This is TNE crude lysate (no detergents).
Add equal volume of 1xTNE +1% SDS, 0.5% NP-40, and 0.5% Doc (Complete TNE).
Sonicate at Temperature40 °C (3 pulses: 10s On/2s Off at 20% amplitude).
Boil (Duration00:10:00 ).


Note
*STOPPING Point: Sample should be in “Complete TNE”. Make sure to sonicate and boil prior storage*.

10m
Centrifuge (Centrifigation16000 x g, 4°C, 00:10:00 ).

10m
Centrifigation
Collect supernatant for BCA protein assay.