I am having issues transferring my large protein (220kDa) at 30V for 2 hours (RT). It is often patchy with incomplete bands (annoyingly I occasionally I get a clear transfer!). For my other protein at around 40kDa (for b actin) I have no issues.
I have read that for larger proteins people either run overnight at 4 degrees at 20V or for 3 hours at 70V.
Ideally I would like to run in the shortest time possible! One of the reasons why is the power pack i use is pretty old and I don't know how well it would withstand the cold room (rusting)!
But my main worry is losing the smaller proteins if I up the voltage for a prolonged time.
I am using pre-cast tris-glycine 4-20% and the transfer buffer is composed of 10% methanol, and tris-glycine buffer (x2) for a wet transfer.
I was wondering what protocols work for you or if you have any suggestions on what I should try first?
Dear Olivia,
I have transfered proteins up to 250kDa and tubulin (around 50kDa) at 100V for 1 hour.
I put the transfer system in ice and I use a precooled transfer buffer ( 20% methanol, and tris-glycine buffer (x1) ).
I hope you find it useful.
Erica
A former lab member of mine had a similar problem.
What he did was put two membranes on top of each other in the transfer sandwich. The smaller ones were transferred to the bottom membrane, while the big protein was left in the upper one. Was a little bit more work with the quantifications later on, but this way he captures proteins which different in size by a factor of 7!
About the settings: We choose the voltage depending on the dimension on our membrane: 0.9*lenght*widht = voltage used.
Hope this helps
Your best option is a slow O/N transfer (30 V, 16 h at 4 or R.T.); robust approach that works for all Mwts, at all percentage gels. You'll get reproducible and strongest possible signals. Other approaches simply don't work well (low recovery) particularly with huge disparities in Mwts.
When I use 100 V for one hour I have the tank immersed in crushed ice with a magnetic stir rod stirring in the tank. Otherwise I transfer overnight in a 4C cold room at 25V. None of my membranes have dried or cracked.
Hi olivia,
I have never had that problem, sorry... have you tried to change the electrophoresis power supply?
Are you controlling the amperage? when you transfer at "X" constant voltage, do the amperage reach the maximum amperage supported by your power supply?
For large proteins, I found the following helpful:
- use tank blotters, not semi-dry. Do use the ice-pack that comes with the unit to keep everything cool.
- use Dunn's buffer (doi:10.1016/0003-2697(86)90207-1, 10 mM NaHCO3, 3 mM Na2CO3, 50 µM SDS), not Towbin's
- use gradient gels (5-10%, with 4% stack), so the big proteins are in a low %T matrix
- for all MWs use PVDF-, not NC-membranes
- after blotting, stain the gel with CBB to check that the proteins have left and the blot with Ponceau to check that they have arrived safely ;-)
- If your protein is glycosylated, try CTAB-PAGE with eastern blot (doi:10.1016/S0003-2697(02)00639-5) for sharper bands
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