HeLa SILAC conditions

Posted on Updated on

Grow cells in DMEM (Sigma #D9443) without L-Arg, L-Leu, L-Lys !

This DMEM is low glucose 1000mg/L so you will need to add and extra 3000mg/L to make it unto 4000mg/L in total. Thus 15ml of the 10% Glucose solution is needed for 500ml of DMEM.

Need to add 50ml of Mass Spec Grade Serum for final [conc.] of 10

1X Normal [Conc.] for DMEM media
L-Arg = 398 µM
L-Leu = 802 µM
L-Lys = 798 µM

From testing HeLa cells grow well and have very high incorporation rate at 1/4 to 1/8 of these levels. To play it safe we have been using 1/4. Thus:

L-Arg = 100µM  (Light and Heavy)
L-Leu = 200µM  (Only Light needed)
L-Lys = 200µM  (Light and Heavy)

Grow cells for at least 5 doublings to ensure complete labelling. As a rough guide, if you Start on Monday, you will be ready by the following monday !

 

 

 

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s