Mar 20, 2024

Protein Lysate Preparation Protocol (Tissue)

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvj3k3xlk5/v1
  • Scott Vermilyea1
  • 1University of Minnesota
  • Team Lee
Jane Balster
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DOI: https://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, protein lysate preparation protocol, preparation of protein lysate, protein lysate, lysate preparation, tissue sample, protein, preparation, tissue
Abstract
This protocol details the preparation of protein lysate of the tissue sample.

Attachments
Icon for Western Blot and Lysate Prep Protocol.docx
Western Blot and Lys...
19KB
Protein Lysate Preparation (Tissue)
4h 20m
If tissue stored at -80 °C , place in -20 °C for at least 04:00:00 or Overnight prior to homogenization.

4h
Weigh tissue out in mg.
Add in 10 volumes of 1X TNE (e.g. 10 µL of TNE per 1 mg of tissue).
Either by mechanical (Dounce) or homogenizer machine, homogenize tissue gently and On 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 40 °C (3 pulses: 10s On/2s Off at 20% amplitude).
Boil (00:10:00 ).


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

10m
Centrifuge (16000 x g, 4°C, 00:10:00 ).

10m
Collect supernatant for BCA protein assay.