I am trying to extract protein from a 3mg tissue. I used 85ul ripa and used a bead homogenizer. It’s a nervous tissue. The protein concentration were satisfactory (using bca) and I proceeded with western but when I did Ponceau staining it looks less protein. Each well is having 50 ug protein.
Top blot:Well 1 next to protein ladder is my concerning tissue and well 5 too! other lanes are heart tissue.
bottom blot: the well next to protein ladder is the nervous ti other labs are heart.
The heart and nervous tissue all had same protein amounts. But I had more tissue weight wise for heart so I added ripa accordingly In those samples when I homogenized.
1- How can I improve protein extraction?
2- what ripa to tissue ratio I shall be using for small tissues?
3- Can fat contamination give me false protein concentrations using bca assay?