2 replies to this topic

[firefly2280]

So sorry this may have been asked before but everyone seems to do something slightly different.

 

We need to extract protein using urea (8M) from S. aureus, avoiding detergent as we will do SDS-PAGE and then LC-MS/MS after. 

 

Ive no idea where to start, need a total of around 1 mg / ml protein but can I just spin down a pellet and add urea and bead beat? Should I add a lysis buffer? 

 

If anyone knows of a rough starting amount of bacteria as well that wil be great but I can work that out if needs be! 

 

thanks

 

 

[neuron]

Starting amount of culture will depend on the yield of your protein. May be you can start up with 500mL culture, lyse the cells and estimate the protein. Then you can back calculate how much culture will be required.

 

You might have to add some buffer that should have protease inhibitors. You can avoid the detergent and do some freeze thaw lysis.

[firefly2280]

Thanks neuron