Hi Anja Angelov

1. What kind of RIPA or other buffer are you using

(I need to isolate all proteins, even those in mitochondria, nucleus, membranes and cytoplasmic, just all of them) - in RIPA are also a lot of options like with or w/o EDTA, glycerol, Triton-X-100 or NP40...)?

I haven't used RIPA but I prepare the lysis buffer using cell lysis buffer from Cell Signaling Technology (10X) #9803 and adding cocktail inhibitor (Sigma), Trypsin inhibitor Trypsin inhibitor (PMSF) 200mM and 0.5% SDS.

2. How do you cut the tissue pieces (liquid nitrogen, cutter) and what amount do you use (10mg, 20mg, 50mg...)?

I prefer to extract protein immediately from fresh heart tissue. Start by cutting about 20-30 mg of heart tissue to small pieces using scissors or blade inside cold PBS buffer to remove blood. Next, transfer the tissue pieces to 150-200 ul of freshly prepared lysis buffer (cold). Then use the homogenizer to get tissue lysate. Keep the lysate on the ice for 20 to 30 minutes (vortex the lysate for few seconds every 10 minutes). Next centrifuge (cold) for 30 minutes at 14000 rpm. Sperate suspension to new tube and measure protein concentration.

3. How long do you incubate the lysation process? I saw everything from 15 minutes til 1 hour... Is it up to the organism (mice, rat, human)?

Its depend on the lysate that you use but I used to keep mouse heart about 20 to 30 min and I vortex the lysate every 10 min).

4. What is your average amount of protein per mg tissue? So what range should I expect?

I think this also depends on the lysation and volume of lysis buffer. But I am sure you can get high concentration if the lysation goes well.

Hope these answers would help

Good luck

Thanks a lot, Isehaq Al-Huseini, for your detailed answers. I prefer also to isolate proteins from fresh tissue - it seems to bee easier because of the cutting problems with hard, frozen tissue. Unfortunately I did not get any but frozen. You cut them praying they are not jumping away from the also frozen underlayer. It is a not that nice work. But....

So also we do not have an homogenizer. I made powder from the tissues with liquid nitrogen and mortar/pestill. After lysation I used a sonificator.

The protocols used do really differ a lot. So the Coomassi staining was wonderful. Will see after antibody incubation if there are my proteins popping up ;-)