Semi-dry system for NuPage with MES buffer - (Oct/01/2013 )
I am using a 4-12% Bis-Tris NuPage (Invitrogen) ready-made gel , and run in MES buffer to enable me to view the protein size of less than 10k Da.
However, when i transferred the gel to the Immobilon PVDF 0.2um using Trans-Blot SD Semi-Dry (Biorad), at 12V for 45 min for the mini gel , the transferring is still incomplete when i stained my gel with coomasie blue after transferring it.
The transfer buffer which I am using is theglyicine , tris base, SDS and methanol.
I did not use the running buffer as suggested for NuPage transfer buffer.
Is it because of the different transfer buffer which causes the inefficiency of the transferring?
yes and no. if you used transfer conditions (voltage, time, current, power) for the nupage buffer with the t-g buffer then yes.
if you used conditions standard for t-g buffer then no.
however, you will need to adjust conditions to improve transfer efficiency. keep in mind that transfer is often incomplete but that is mostly with high molecular weight proteins (with low mw proteins you would probably need to silver stain to see the remaining protein).
how much sds and methanol do you have in your transfer buffer? what are the 1x concentrations of tris and glycine?
if the concentrations are too high then migration may be slowed.
Thanks alot for the reply.
I am using a 20% methanol + 0.02% of SDS in my transfer buffer, which is a 1X tris and glycine.
you can increase the sds to 0.05% (some literature says not to exceed this concentration, others say 0.1%, i adhere to 0.05%).
the pI of your protein may be too close to the buffer pH to migrate quickly.
here is a link to ge's handbook page (you can download their western blotting handbook, as well as other useful handbooks).
i'm attaching a pdf of millipore's handbook and troubleshooting guide for protein blotting.
Thanks alot for the advices and is really nice of you to share the handbook and link.
I will try again.
Have a nice day :D