My protein of interest is a bacterial inner membrane associated protein. And I suspect it to be forming an insoluble aggregate in a mutant strain. I read that Blue Native PAGE works better than just Native PAGE for membrane proteins. Is that true? I was planning on running a Blue-Native PAGE and then doing a Western with it to test if my protein forms an aggregate. Alternatively, is it better to just run a Native PAGE here instead of Blue Native PAGE?
I appreciate in advance all your help/guidance/comments addressing my question.
PS: We have a BIO-RAD mini gel apparatus in the lab, so I would love to have a protocol that works with this system.
Blue Native PAGE uses Coomossie-G250 to give proteins and protein complexes a negative charge so the proteins move towards the Anode(+), kind of like they do in SDS PAGE ... except SDS linearizes proteins and breaks apart protein complexes,
but Blue Native PAGE preserves protein complexes as negatively charged.
If you just do Native PAGE if your protein or protein complex is positively charged then it will move towards the cathode(-) and hence never enter the gel. If either is neutrally charged then there will be no movement.
If both are positively charged at your buffer pH then you can just switch the electrodes going to the battery (black to red and red to black) and do Native PAGE and hence the proteins and protein complexes will move down the gel towards what is now the cathode(-) not the anode anymore.
- Badges
- Science topic
- Similar topics
- Biological Science
- Kinesiology
- Running