protein extraction from cell pallets
Jacob Waldbauer, Lichun Zhang
Published: 2022-01-25 DOI: 10.17504/protocols.io.drh535
Abstract
Use LDS buffer to extract protein from cell palllets
Before start
Steps
1.
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.
2.
make 1M DTT stock solution in LC/MS water.
3.
Add 200~500uL of 10mM DTT in 1X LDS buffer to the cell pallet tube.
4.
Heat sample up at 95ºC for 20 minutes.
5.
Cool down the sample to room temrature. Meanwhile, make a 0.5M iodoacetamide stock solution.
6.
Add IAM to 50mM/60mM in the sample, incubate in dark for 30-60minutes.
7.
Add DTT to 50mM/60mM. Sampe is ready to continue with FASP.
