What is your lysis buffer? may it is not stringent enough to lyse and extract proteins efficiently from cell?

I had a similar problem some time ago and I came up with the following solution,

- Plate your astrocytes on 100mm Petri dishes

- Use about 700 μl-1 ml of lysis buffer (RIPA) and scrub your cells

 Now you need to get rid of the excess of solvent to concentrate your proteins, I can propose two ways to do it:

1. Use centrifugal filters (I used Ultracel – 3K Amicon Millipore) add 3 ml of PBS to your lysate and centrifuge it down to about 100 µl, you can repeat this step once again

2. Freeze your lysate in - 80°C and lyophilise it. Than just resuspend your lysate in a desired volume (In this the approach the excess of salts from lyssis buffer might cause blurred blots, but it is not a rule and you need to test if it works for you).

In both methods you should get about 3 µg of proteins per µl

Add 500ul to 1ml 2x sample buffer, Scrap cell off the plate using a sterile cell scrapper, aspirate using pipette into a 1.5ml eppy tube, then homogenize using a sterile 5ml syringe with a 22 gauge needle, store as aliquots in -80C.