Fast Westerns

[gosens08]

Does anyone have ideas on how to speed up Westerns and the concerns associated with them?

Can you reduce the transfer speed to 20 mins or under by running at high voltage? Or can you reduce/eliminate blocking time? I mean, if the obviously do not work then you have to retry without the shortcuts, but are there other problems?

[danfive]

I do semi-dry transfer in 20 minutes and am doing very well without blocking steps.

[trypsin]

@danfive
what kind of gels do you have? size? How many current do you use in this 20minutes?
trypsin

[admin]

lol that is a good question gosen,

I actually did my western blotting and transfers in less than 1 hour due to some short cuts. Note do not try this with tough westerns or phospho-proteins.

1) quick transfer in cold room with ice packs and high voltage 20 minutes
2) quick primary 10-20 minute incubation with 5% blotto milk to already start blocking!
3) quick washing with LOTS of wash buffer each time - make sure to get rid of all before you add fresh
4) quick secondary 10 min (with milk blotto 5%)
5) quick washing with LOTS of wash buffer each time make sure to get rid of all before you add fresh
6) RESULTS!

(dont try this at home lol and also dont tell my previous supervisor lol)

[danfive]

Originally Posted by trypsin

@danfive
what kind of gels do you have? size? How many current do you use in this 20minutes?
trypsin

4-20% Tris-Cl (biorad readygels). Minigels-~7cm long. 17V, 0.50mA(limit), 19min.

[pabells]

blotting will depend on the size also of you protein of interest. Lower MW will transfer faster than larger.
If you're interested in speeding up the steps of blocking membranes, primary and secondary incubations, and washing. There are faster protocols that are being developed by Pierce-Thermo Fisher scientific. piercenet.com

[radioheadhead]

check out the SNAP ID gizmo on millipore.com (just search the term).

I did a complete WB in four hours, including electrophoresis, transfer, and blotting/ detection. This thing is awesome.