Protein Lysate Preparation Protocol (Culture)
Scott Vermilyea
Published: 2024-03-21 DOI: 10.17504/protocols.io.e6nvwdwd9lmk/v1
Abstract
This protocol details about the protein lysate of the culture.
Attachments
Steps
Protein Lysate Preparation (Culture)
1.
Wash cells using cold PBS followed by ice-cold 1x TNE lysis buffer containing 1% SDS, 0.5% NP-40, 0.5% DOC and protease/phosphatase inhibitors.
TNE lysis buffer:
| A | B |
|---|---|
| Tris | 50 mM |
| NaCl | 150 mM |
| EDTA pH at 7.4 | 5 mM |
2.
Use cell scraper to remove all detached cells and keep On ice for 0h 5m 0s.
3.
Transfer lysates to 1.7mL collection tubes and sonicate for 2 pulses (at 20% amplitude, 0h 0m 10seach), heat for 0h 10m 0s on hot plate (100°C) and centrifuge at 16000x g,4°C.
4.
Transfer resulting supernatants to new tubes (final total lysate).
5.
Perform BCA protein assay following manufacturers instruction and prepare 1xLaemlli samples of standardized concentration for SDS-PAGE.
