Western blotting conditioned media problem - Media will not separate when run on gel (Feb/27/2008 )
I am having a lot of trouble doing western blots on conditioned cell media. I am using media from CHM5 cells (microglia) and CCF-STTG1 (astrocytes). When I run this media on a 10 or even 15% acrylamide SDS gel I only get one big band at ~75kDa. Does anyone know how I can separate out my media? I have tried adding more BME to the loading buffer, I spin my samples for 3 mins before adding loading buffer and denaturing for 5 mins at 95 degrees. I need to look at the secretion levels of a cytokine at 25 or 8.7kDa. Thank you in advance.
Sounds like a cross-reactivity problem, Do you use a serum in your medium? It will affect the results you get for a western, unless it is washed out of samples (I know you aren't using whole cell lysates, but it is worth mentioning anyway). Have you tried exposing your images for a long time, ignoring the bright band, just to see if you can see your cytokines?
I'm not familiar with your cells and culture condition, so my first question is: do you use serum in your media? If yes, the band can be albumin (BSA is ~60 kDa, if I remember well, I don't know how accurate your size is).
On option that comes to my mind is to do a concentration of your samples using a 30 or 50 kDa cut-off MW (Millipore or Vivaspin). This will not eliminate completely your abundant protein but can decrease it so that there is less non-specific reaction with it.
I hope this helps or a better insider comes soon and gives you a good idea.