Hi everyone,

I am currently trying to resolve a 500kDa protein, but to no avail. I am using a 7.5% polyacrylamide gel in a 1X Tris glycine buffer.

Moreover, I can't seem to find a protein ladder suitable for a molecular weight of this size. NuPage makes one called the HiMark, but its for tris-acetate gels (ones only they supply)

Any suggestions??

Thanks

Whoa! My largest has been ~400 (PC1) - best advice: 4% gel - handle it very gently for transfer, silanize (RainX works) plates, place membrane on gel (not the other way around), and wet transfer overnight - I'll check and let you know the voltages).
Also - is this a native gel, or a denaturing/non-denaturing SDS?

For the marker, thyroglobulin may be a good choice. You may need to add that to a std commercial marker (max ~200-220 kDa). Antibodies to bovine thyroglobulin are also available if you want to use it as a protein loading ctrl.

For the overnight (~13-14 h) transfer,
a) use chilled buffers, and do the transfer in a cold room
b) for a mini-~8-12 cm gel (I'd used the BioRad miniProtean2 most often, but anything similar will do),
30 V transfer setting
(constant voltage: usually ~90mA initial current).
I'd used both denaturing and non-denaturing SDS PA gels - its different for native gels, which is why I asked.
You might also try out something like Amresco's 5% self forming gradient NEXTGel (ISCBioexpress) - fairly cheap (~$26 for 10 gels rgts - all you need is APS and TEMED) - they are a little less fragile than the 4% gels, but still require VERY careful handling.
All the best, and let us know when you manage to tame it - including any tweaks!