Protein from Liver tissue - (Jun/13/2007 )
Hi, has anyone experience with extraction of proteins from Liver tissue? I want to do Western Blots, but every time I try I get nasty prezipitations when denaturing them, and even when i do not heat them up at all. The prezipitate is yellowish and my ß-actin Signal is very low. With Colon Tissue I don´t have this problem. 
The protein isolation is just the normal: Buffer and inhibitors on it, disrupting with Homogenisator and zentrifuge, done. I´m the only one doing Westerns in the whole house and its my first time. I wanna show a good overview over the main organs, so is there anything to consider in some organs? (If you have information about other organs, plz write them down, too. I don´t know what I´m doing here and there´s noone to ask)
The buffer contains NaCl, HEPES, EDTA and NP40, pH 7.4 + fresh Inhibitors Pepstatin, Leupeptin
the loading buffer contains Tris, SDS, GLycerol, Bromphenolblue and ß- mercaptoethanol
i would suggest to add more 5X buffer to your sample.
When it happens to e, i dilute the sample with running buufer (tris glycine SDS).
The protein isolation is just the normal: Buffer and inhibitors on it, disrupting with Homogenisator and zentrifuge, done. I´m the only one doing Westerns in the whole house and its my first time. I wanna show a good overview over the main organs, so is there anything to consider in some organs? (If you have information about other organs, plz write them down, too. I don´t know what I´m doing here and there´s noone to ask)The buffer contains NaCl, HEPES, EDTA and NP40, pH 7.4 + fresh Inhibitors Pepstatin, Leupeptin
the loading buffer contains Tris, SDS, GLycerol, Bromphenolblue and ß- mercaptoethanol
I extract proteins from spleen, heart, liver, kidney and lung. I work with mouse organs, therefore I can give you only information about my experience with them. Used amounts: e.g 1-2 liver lobes.
For each of the organs I try to estimate the amount of lysis buffer (normally use RIPA buffer, you can find the recipe elsewhere). First of all I homogenize my tissue in PBS/Protease Inhibitor cocktail. After zentrifugation i resuspend the pellet in approx. 400µl RIPA lysis buffer 30min (vortex every 5-10 min). Again zentrifugation, now you have whole cell extracts. To improve the amount you can repeat this lysis step with the pellet you get from the zentrifugation before. And pool the supernatant of the 1st and the 2nd lysis. In my case it worked very well.
good luck