Dear Ulrika, although we do not work with brain tissue (yet), I can give you a hint on the protein yield we have with fat tissue that also contains a substantial amount of lipids. On average, after lipid extraction, our protein yield is around1.5- 2 mg per 100 mg of tissue.

Thank you Jonah for your hint! I probably should redo the measurements because I got around 14 mg/ml (the volume is around 200 ul) which seems a bit to good to be true. Mostly I got 10 x less, therefor this question...

Maybe you have some interfering substances in your sample that are not compatible with your protein assay. Particularly lipids and the BCA assay is a no go. Make also sure that your protein assay can handle the buffer you used to dissolve the proteins. 

That's why I did the centrifugation after homogenization in order to get rid of the lipids in the sample (the supernatant was kept, the lipids/cell debris was thrown away)...what else would you suggest?

RIPA is a widely used lysing buffer. But that is true that I couldn't used it for the BSA standards because it was interfering with the BCA working reagent (wasn't really changing the color normally). So I understood that if RIPA is in larger amounts/higher concentrations it does interfere with the assay.  I used RIPA only as the blank which optical density number was subtracted from the sample optical density number afterwords to get the real numbers.

Thank you Johan for your comments though! 

Hey there,

I had same situation when I've started the WB. Have you tried to prepare new lyse buffer again and to use more protease inhibitor? and I do not know whether you had or not but you also could add the protease inhibitor to lyse buffer each time before using.

Dear Ulrika, I'm not sure whether centrifugation alone will remove all of the lipids. In our situation with adipose tissue this certainly leaves quite some lipids. That is the reason why I apply a lipid extraction (Chloroform/Methanol works well) during my protein isolation procedure. What we see is that without lipid extraction we can identify roughly 300 proteins with LC-MSMS while with a lipid extraction this is almost doubled. Your method of data extraction may indeed explain high protein concentrations. For proper determination of your protein concentrations the standard should be prepared in the same buffer as your samples are in. As an alternative you may check the DC protein assay from BioRad. Good luck!

@Turan I use a commercial available RIPA buffer and always fresh protease inhibitor cocktail. To use more protease inhibitors? Well, I could add more if you think that it helps...

@Johan, interesting...but why are all this papers published with no additional lipid extraction? I know some colleagues who are not even doing the centrifugation part (they just homogenize the brain structure with lysis buffer and protease inhibitors and denaturate them with Laemmli buffer). Thank you for your input! I definitely will investigate this issue further...

Dear Ulrika, I referred to adipose tissue. We had a problem of not detecting a protein of interest, which popped up with the lipid extraction method. Apparently, in previous work, as published, proteins of interest were more easily detected. 

Hi Beitnere, we usually probe sonicate mouse cortex/hippocampus in 5 to 10 volumes of PBS + TritonX100 (0.5%) and protease inhibitors (Roche EDTA free). We normally get about 1 - 2 mg/mL in the soluble phase after a 16,000 g spin (4C, 15 min). I dilute my samples 1:10 for BCA assay and use BSA standards prepared in 1:10 diluted extraction buffer. I have only tried RIPA a few times but it yielded similar results. There will still be lipids in the supernatant as Johan stated (some microvesicles as well as lipidated proteins).