Jan 25, 2022
protein extraction from cell pallets
- Jacob Waldbauer1,
- Lichun Zhang1
- 1University of Chicago
- Coleman Lab
Protocol Citation: Jacob Waldbauer, Lichun Zhang 2022. protein extraction from cell pallets. protocols.io https://dx.doi.org/10.17504/protocols.io.drh535
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: September 04, 2015
Last Modified: January 25, 2022
Protocol Integer ID: 1545
Abstract
Use LDS buffer to extract protein from cell palllets
Make 1X LDS buffer. In 40mL of 1X buffer, add Tris HCl 0.666g, Tris base 0.682g, LDS 0.8g, EDTA 0.006g, Glycerol 4g.
make 1M DTT stock solution in LC/MS water.
Add 200~500uL of 10mM DTT in 1X LDS buffer to the cell pallet tube.
Heat sample up at 95ºC for 20 minutes.
Cool down the sample to room temrature. Meanwhile, make a 0.5M iodoacetamide stock solution.
Add IAM to 50mM/60mM in the sample, incubate in dark for 30-60minutes.
Add DTT to 50mM/60mM. Sampe is ready to continue with FASP.