Native Page transfer/detection problems - (Oct/22/2009 )
I am having issues with native page. I am using the NativePAGE protcol/reagents from Invitrogen - blue native page - to try and assess the dimer:monomer ratio of my protein of interest. Running the samples seems fine, but when I transfer to PVDF the membrane is quite blue. I fix the proteins with 8% acetic acid, rinse and air dry, then rewet with methanol, but this does not completely remove the dye. When I eventually go to scan the membrane (using the Odyssey infrared system) the dye shows up everywhere and I have no idea what is what. So I guess I would like to know:
1. What do I need to do to in order to completely remove the Coomassie dye?
2. Do I need to use blue native page in order to determine the dimer:monomer ratio or can I just not add SDS and not heat my samples and then run as I normally would for denatured samples? I have seen some papers that have done this, at least according to what they have written.
3. Can I just stain the gel and then quantify without worrying about transferring and using antibodies etc?
I have never run a native page before so any help would be great.
you will never get all the dye off of the membrane. you can use a chemiluminescent or fluorescent detection system to visualize your result.
there are or methods for native page besides blue native. you can use a method like ornstein-davis or some other (the protocols for some other native methods should be somewhere in the archives of this forum, if not, then i can re-post some).
staining the gel should be fine if you know for which bands you are looking.