Hello
I’m trying to make western blot samples with human blood serum. But when I mix the serum with 5x sample buffer and boil it, the sample aggregates and becomes unable to load.
So I used EDTA to the serum. It wouldn’t harden, but when I loaded the sample and proceeded western blotting. it became all blurry and messy.
does anyone know how to deal with this problem?
Harman
Blood Serum already has soluble proteins and it shouldn’t form any aggregates. I have worked on blood plasma proteins for over 20 years and never faced this issue. Probably your sample buffer has low concentration of BME or DTT & SDS. In case of Plasma, fibrin clots may form sometimes but that can be removed by Centrifugation. I suggest preparing fresh sample buffer. Regarding the smear on blot, if you are loading raw serum, then salt concentration (>150mM) in serum can interfere with resolution and high protein content too. I suggest that you first remove salt by using Acetone/TCA precipitation followed with SDS PAGE. Dont load in access of 75 to 100 Microgram Protein.
Let me know if that doesn’t help!