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optimal conditions for wet transfer in western blotting - (Nov/18/2008 )

dear all
I will be shifting from semi dry transfer to wet transfer in western blotting. I would greatly appreciate if you provide me with info regarding optimal conditions (% of methanol in transfer buffer , ampere, and time) for wet transfer that help me detect both large (about 150kDa) and small (about 15 kDa) molecular weight proteins separated on 10% and 12.5% SDS-PAGE gels.
comments are welcome
thanks

-yobou-

QUOTE (yobou @ Nov 18 2008, 09:16 PM)
dear all
I will be shifting from semi dry transfer to wet transfer in western blotting. I would greatly appreciate if you provide me with info regarding optimal conditions (% of methanol in transfer buffer , ampere, and time) for wet transfer that help me detect both large (about 150kDa) and small (about 15 kDa) molecular weight proteins separated on 10% and 12.5% SDS-PAGE gels.
comments are welcome
thanks


why go for wet transfer? most people use semi dry. it consumes less transfer buffer.

-Curtis-

QUOTE (yobou @ Nov 18 2008, 10:16 PM)
dear all
I will be shifting from semi dry transfer to wet transfer in western blotting. I would greatly appreciate if you provide me with info regarding optimal conditions (% of methanol in transfer buffer , ampere, and time) for wet transfer that help me detect both large (about 150kDa) and small (about 15 kDa) molecular weight proteins separated on 10% and 12.5% SDS-PAGE gels.
comments are welcome
thanks



Hello,

Transfer buffer we use is:
800 mL Water
200 mL Methanol

Then add:
5.6 g Tris-Base
3 g Glycine

Transfer time fluctuates depending on the target proteins.

For 10-25 kDa proteins i transfer for 70 min
For 25-50 kDa proteins i transfer for 85 min
For >50 kDa proteins i transfer for 90 min

All done at 65 volts. I routinely use 10-12% gels.

*Also for 150 kDa proteins, i'd suggest you change the gel concentration down to 5 or 7%. Anything over 50/60 kDa are sometimes hard to visualize on 10-12%'s.

-Bbop-

you may also want to add 0.05% sds to the transfer buffer for the 150kDa protein to facilitate the transfer.

-mdfenko-