I've already tried this hApoB100 - it is almost 500kDa- western blot 3 times. My sample proteins are from mouse cortex and hippocampus.
So, I run on 4-12 % gradient gel and load 100ng protein into each well, and ladder (10-245kDa). The running time was 3 hour,s RT, 50, then 100mV. Transfer (wet) was more than 3 hours, in 1x transfer buffer (No SDS added). I used nitrocellulose membrane, charged in methanol. Blocking with 5% milk-PBS, RT, 1 hour. Antibody was hApoB100 (It should interact with ApoB48 and 100), 1:200, overnight in 4C°. It was diluted in azide-PBST. Secondary antibody was policlonal rabbitxgoat , 1:35000, 45 min, RT.
But there is no band on the rtg film, just a huge mass on the lane of the purified protein. On the membrane, after Ponceou staining, are no sign bands after 200 kDa.
Should I try with 0,1% SDS transfer buffer? Or longer transfer time?
Thanks for helping!
WESTERN BLOT - high molecular weight protein