Hi everyone,
I am working on oxidative stress induced Peroxiredoxin 2 dimerization. I have been using the microbiospin filtration to remove excessive DTT. My protein gets easily oxidised so, I treated the column with approximatively 10ug/ul of catalase to remove H2O2. Then I pre-equilibrated the column using sodium acetate pH 4.5 (3 washes).
Pre-equilibration process: after removing excessive buffer (50mM tris), I applied 500ul of sodium acetate, mixed it up and down several times using a P1000 before removing excessive buffer. I then applied 50ul of my sample. After running electrophoresis followed by silver staining. I noticed that most of the sample was lost.
Do you know what could have hindered Micro-biopsin column purification?