I am doing protein pulldowns with biotinylated RNA baits. I start with a lysate of approximately 20x106 cells (one 150mm dish close to confluency). First I bind the biotinylated RNA molecule to streptavidin magnetic beads (Dynabeads™MyOne™ Streptavidin T1). I have been using 1mg for each 150mm dish. Following the beads' manual, I add 400pmol of the biotinylated molecule per mg of beads. After the RNA is bound, I incubate with the lysate. I started incubating for 30 mins @ RT and have been increasing to 2h @ RT and ON @ 4C. I finally elute by boiling 10 mins in 100uL of Laemmli Buffer.

The issue is that I need these samples to identify the bound proteins by mass spectrometry and the final concentration after elution is very low, generally around 0.4-0.25mg/mL. In our MS facility, they require 50uL of a sample at least at 1mg/mL. The concentration of the initial lysate is around 3-5mg/mL generally.

Can anyone share his/her experience? Any protocols?

Is it possible to improve protein recovery without affecting the specificity of the pulldown? 

Do you think I need to increase the initial number of cells any further?

Thank you very much in advance!