GM1 detection on blotted membrane - (Jul/24/2006 )

I'm analyzing GM1 ganglioside in lipid raft. Now I tried to detect GM1 on blotted membrane using choleratoxin B-subunit-HRP, but I could get no signal. Please anyone tell me the protocol for detection GM1?

Thanks

-------Protocol in brief--------
Sucrose density gradient centrifugation& recover fractions->SDS-PAGE(10% gel) (GM1 would migrate with the dye front)->electro-transfer to nitrocellulose (or PVDF) membrane ->blocking with 4% skimmilk for 30min-> incubate with Ctx-HRP in 4% skimmilk for 1h->wash PBS-Tween x 3times->Detection with ECL

-hbj-

One possibility: milk or Tw80 washed away your GM1 lipid, although there could be quite a few other reasons as well. You may have to use less harsh condition to block and wash your membrane.

-genehunter-1-

Thanks for your answer, but when I detected GM1 by Dot blot (Samples were bloted directly and detedted described above), I could detected it. Therefore there would be another reason.

QUOTE (genehunter-1 @ Jul 25 2006, 03:52 PM)

One possibility: milk or Tw80 washed away your GM1 lipid, although there could be quite a few other reasons as well. You may have to use less harsh condition to block and wash your membrane.

-hbj-