Hi everyone!
I am trying to detect ATM expression in Hacat cells through western-blot. ATM is a large protein (350kDa) and I have tried running 60ug of lysate - 6% polyacrylamide gel for 3.5h and transferring overnight (nitrocellulose membrane) at 20V and I could not see any signal so far. Do you have any recommendations on how should I proceed?
I do not use 0.1% SDS or methanol in my transfer buffer.
I appreciate any input you may have.
Run Disc SDS PAGE with 4% stacking and 7% Resolving gel. Once run is complete, electroblot on NC member and check transfer of protein with Ponseau S dye. Once transfer of 350 kDa protein is confirmed, probe using an appropriate anyibody-enzyme conjugate.
You can do the electrophoresis in gels of low %T, if you stabilise the gel with 0.5% agarose (electrophoresis grade, see doi:10.1006/abio.1995.1004, 10.1007/978-1-4939-8793-1\_18). Agarose forms very large pores and therefore does not influence the electrophoresis result.
For blotting, I'd suggest Dunn's buffer (doi:10.1016/0003-2697(86)90207-1, 10 mM NaHCO3, 3 mM Na2CO3, 50 µM SDS), which is not only cheap, but also works with large proteins (although I have tested it only up to 160 kDa). Methanol is counterproductive, it strengthens binding of small proteins to membranes, but prevents mobilisation of large proteins from the gel.
If you want to save yourself a lot of grief, use PVDF membranes, not NC. They not only have higher protein binding capacity and affinity, they are also mechanically and chemically stronger.
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