Jul 21, 2025

Public workspaceMouse tissue lysate preparation

DOI
https://dx.doi.org/10.17504/protocols.io.14egnyd3zv5d/v1
  • Hely Rodriguez Cruz1,
  • Michael Hanna1,
  • Pietro De Camilli1
  • 1Yale University
Michael G Hanna
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DOI: https://dx.doi.org/10.17504/protocols.io.14egnyd3zv5d/v1
Protocol Citation: Hely Rodriguez Cruz, Michael Hanna, Pietro De Camilli 2025. Mouse tissue lysate preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnyd3zv5d/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: June 05, 2025
Last Modified: July 21, 2025
Protocol Integer ID: 219603
Keywords: ASAPCRN, mouse tissue lysate preparation this protocol, preparation of lysate, lysate preparation, mouse tissue, lysate, detergent, preparation
Abstract
This protocol is for the preparation of lysate of mouse tissues using a detergent in non-denaturing conditions
Materials
  • Ice
  • Mouse tissue
  • 10-15mL dounce homogenizer
  • MilliQ H20
  • 15mL conical tubes
  • 1.5mL microcentrifuge tubes
  • TRIS
  • SDS
  • NaCl
  • Protease Inhibitor Cocktail
  • Heat Block
Troubleshooting
Mouse tissue lysate preparation
Weigh tissue, keep on ice, and prepare 1mL of Homogenization Buffer for approximately every 100mg of tissue.

Homogenization Buffer:
  • 25mM TRIS pH 7.5
  • 150mM NaCl
  • 1x Protease Inhibitor Cocktail
Using a tissue homogenizer, submerge your tissue in the appropriate amount of homogenization buffer (1mL/100mg) in the homogenizing vessel. Homogenize the tissue thoroughly. Be sure to keep the tissue grinder submerged while homogenizing to avoid frothing.
Transfer the homogenate to a 15mL conical tube and add 0.5ul of benzonase per mL of homogenate.
Using a concentrated stock solution of SDS, bring the homogenate up to 2% SDS. Use the equation below to determine the volume of SDS to add from your stock solution to your homogenate in order to bring it to 2%. The volume of stock solution to add is as follows:

Vstock = Vstart / ([stock]/[target] - 1)

where Vstock is the volume of stock solution to add, Vstart is the total amount of homogenate you start with, [stock] is the concentration of your stock solution, and [target] is your final desired or target concentration (in this case 2%)

Take a small sample for BCA analysis (plan to dilute ~5x for BCA).
Add concentrated loading sample buffer and bring to 1x. Aliquot the lysate into microcentrifuge tubes. Boil tubes @ 95°C for 3 minutes.
Spin samples briefly at max speed for 10 seconds to spin down condensation left on the caps. Load samples immediately in a gel or store @ -20°C.