Cell Lysates For Western Blotting

 

<center> <h1> Cell Lysates For Western Blotting</h1> </center>

Reagents / Solutions

Lysis Buffer:
10ml 10% Sodium dodecyl sulphate (SDS)
10ml Glycerol
10ml b-mercaptoethanol
8ml 0.5M Tris pH6.8
1ml 0.1% bromophenol blue
51ml H₂ O

Protocol

1. Spin down 10⁶ cells and wash, if required, in phosphate buffered saline.
2. Resuspend cells in 100µl warmed lysis buffer, vortex then boil for 5 - 10 minutes to break up DNA.
3. Cool (not on ice - the SDS will precipitate) and centrifuge to collect droplets.
4. Vortex briefly to mix.
5. The samples may now be analysed by SDS - PAGE and Western Blotting , or
6. Store samples at -20°C until required.