WESTERN BLOT - high molecular weight protein

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ndora
ndora's picture
WESTERN BLOT - high molecular weight protein

Hi!
 
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!

Chin Fen Teo
Chin Fen Teo's picture
Hi ndora,

Hi ndora,

If you only loaded 100ng of cortical and hippocampal lysates per lane, I suggest you to go higher amount than that. Says, 20 to 30ug.

Also, for transferring a protein that large, you may want to transfer for longer time- 20 to 30 V for 16 hours at 4 degree.

Another thing is to drop your methanol down to 5%. And yes, you can also add SDS, but I won't suggest you to go higher than 0.05%.

Good luck.