Blue Native PAGE - Problems with westerns of Blue Native Gels (Jan/15/2011 )
I am hoping that someone out there can assist me with a problem of running westerns from Blue Native Gels. Basically, I am not able to transfer low molecular weight proteins in my samples (and MW markers), which I have been attributing to the extended and profound dye front (from bottom of the run to 200 kDa, to some extent). I have been using Invitrogen’s setup and protocol. I am running Novex 3-12% Bis-Tris gels and have been loading my samples with 5% G-250 and have been running the electrophoresis with a cathode buffer containing .02% G-250 for 1/3 of the gel, and then switching to a buffer with .002% G-250. When I probe the PVDF membrane with my antibody, I am detecting higher molecular weight complexes that are immunoreactive, but I can’t detect a band corresponding to the monomer MW of my protein because it is in the problematic range (~78 kDa). I have tried several fixes recommended by the manufacturer to no avail.
1) Running the gel longer (helps a little), 2) only using the .002% cathode buffer, 3) trying Novex 4-16% Bis-Tris gels, 4) changing several transfer parameters & 5) several attempts using (or not) acetic acid to fix the proteins on the membrane prior to rinsing of the dye with methanol. I would greatly appreciate any thoughts you people might have. Thank you in advance, Greg – desperate postdoc
Postdoc Greg on Sat Jan 15 17:40:17 2011 said:
I am hoping that someone out there can assist me with a problem of running westerns from Blue Native Gels. Basically, I am not able to transfer low molecular weight proteins in my samples (and MW markers), which I have been attributing to the extended and profound dye front (from bottom of the run to 200 kDa, to some extent). I have been using Invitrogen's setup and protocol. I am running Novex 3-12% Bis-Tris gels and have been loading my samples with 5% G-250 and have been running the electrophoresis with a cathode buffer containing .02% G-250 for 1/3 of the gel, and then switching to a buffer with .002% G-250. When I probe the PVDF membrane with my antibody, I am detecting higher molecular weight complexes that are immunoreactive, but I can't detect a band corresponding to the monomer MW of my protein because it is in the problematic range (~78 kDa). I have tried several fixes recommended by the manufacturer to no avail.
1) Running the gel longer (helps a little), 2) only using the .002% cathode buffer, 3) trying Novex 4-16% Bis-Tris gels, 4) changing several transfer parameters & 5) several attempts using (or not) acetic acid to fix the proteins on the membrane prior to rinsing of the dye with methanol. I would greatly appreciate any thoughts you people might have. Thank you in advance, Greg – desperate postdoc
What do you mean 'not be able to transfer'? Are the low MW markers still in the gel or are they just not visible on the blot.
You could be overtransferring. Smaller proteins transfer faster than larger ones. If you have the Amps to high during your electroblotting for too long of a time, the proteins can actually get onto the membrane and then continue until they go right through the other side.
If this is happening (your lower MW ladder just isn't on the blot and it's not on the gel either), then you can try doubling up your nitrocellulose to test if this is what is occuring. If you have overtransfer, you should be able to see your low MW ladder on the most underneat blot, with the higher MW ladder on the blot that is in direct contact with the gel. You could use a lower amps/volts during the electroblotting (or a shorter time) or add SDS to the transfer buffer (I know you can do this with mini-gels/blots, but you may not with your size gels/blots).
Of course, I could have misinterperted your question.
Hi Greg,
I suspect your G-250 is either blocking the transfer or blocked your antibody binding site, or could be both. Try wash away by using tween-20 (sorry could remember which paper mentioned that at the moment)... or wash it away with methanol (could be a risk there for your future assay...)
Just my 2 cents.
Hi Greg,
If you think it is the G-250 interfering at some point some things you can try are:
Leave the G-250 out of the sample prep buffer completely. This is what I do, as my protein of interest is not membrane bound and I don't use any detergents. I also drive my samples through a 28G syringe needle 10 times to achieve lysis.
Replace the 'dark blue cathode buffer' with the 'light blue cathode buffer' earlier. I run my 4-16% gels 150V, 15min dark, then 150V 90min light.
Alex