Greetings,
I've performed this protocol using many samples, however, I've never seen anything like this kind of pellet. My hypothesis is that the protein concentración is very high, but I can't prove it since my luck has ran out and won't be able to "cook" Bradford until friday (H3PO4 is still on it's way to our lab). The extraction was performed using:
-50 uL of sample + 400 uL methanol (vortex).
-Added 100 uL chloroform (vortex).
-Added 300 uL Milli-Q water (vortex.
-Spin 1 min at 14000 x g.
-Added 400 uL methanol (vortex).
-Spin 4 minutes ay 14000 x g.
This huge mass came out just at the interface after first spin, and then at the bottom like any protein pellet should. I'll thank you very much for any insight You could have about this issue.
Regards
Adán
These are the "Pellets" I obtained, they are 2 different IMAC fractions, 100 and 150 mM imidazole. They where purified using HisTrap FF, PBS (500 mM NaCl) and the sample was clarified expi293 expression Medium (25 mL).
Wells, if this is helpful to anyone, I still don't know the real reason for this to happen. Anyway I managed to work things out by changing methanol, so contaminated methanol it is. I don't know about what kind of contaminant, but this allows me to sleep at nights.
- Badges
- Science topic
- Similar topics
- Thesis Research