i am planing to look at alpha glycosides but I I have PVDF membrane and I will use semi-dry method but how I should do western blot and how prepare blocking buffer after running the membrane in semi-dry
please send me protocol or answer please In some way
Mun Wah Woo
Hi Shoxan,
Check our the Proteintech Group institutional posts, we've added a complete guide to western blot and some other common antibody applications. Hope it helps.
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Engelbert Buxbaum
- At least for larger proteins (I work in the range 80-160 kDa) I'd not use semi-dry, but tank blotters. They work better, presumably because the heat produced by the current is distributed to a larger volume.
- Use Dunn's transfer buffer (doi:10.1016/0003-2697(86)90207-1, 10 mM NaHCO3, 3 mM Na2CO3 plus 100 µM SDS for larger proteins), it is cheaper and works better than Towbin's.
- All incubations are in Blotto, for blocking use 5% milk powder in Blotto for 15 min (longer only reduces sensitivity)
- Meth. Mol. Biol. 1312 (2015) contains detailed recipes for blotting.
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