Trouble Western Blotting a 360kDa Protein - (Apr/09/2012 )

Hello,

I have been trying for weeks to western blot a 360kDa membrane protein. I've followed suggestions from online posts but I haven't been successful.

I have prepared 3 different lysates (Thermo Scientific M-per and Mem-Per kits and one using RIPA buffer) using three different cell lines (Two of which should have this protein of interest).

I am currently using a 4-15% gradient gel, I load about 60 ug of my samples, run the gel at 150V for 2.5 hrs, transfer onto nitrocellulose membrane over night in tris-glycine based buffer with 0.05% SDS and 20% methanol overnight at 30V and then for 1 more hr at 70V. (I've also tried other transfer times and voltages as well as transfer buffer with only 0.037% SDS)

Staining of my membrane after transfer with Ponceau stain does not show bands at the expected location but I have also stained my gel once after transfer and no bands are present at the expected location either.

Can anyone please offer any suggestions?? I am at a dead end.

Sounds like you're doing everything just fine. I used to do Westerns for DNAPK (450kDa), and I would just run a 7 or 8% gel and then transfer a bit longer and reduce methanol to 10%.

Were you using Criterion TGX 4-15% precast gel from Bio-rad? If this is the case, the gel will only separate proteins range from 20-250 kD optimally. As mentioned by doxorubicin, reducing methanol to 10% is good to try.

Try running a tris acetate gel which resolves really large proteins well

Ponceau may not be sensitive enough to detect a band in the desired location - try probing with an antibody.

This may be a stupid suggestion- but if you are using nitrocellulose you may want to check your pore size. I know that the nitroceullose commonly used at our institution is pore size 0.45um, which allows proteins over about 110kDa to blow through during transfer (in my experience). Try a 0.2um pore size, or PVDF (I personally have much better results with PVDF for larger proteins).