Western Blot of Large Protein Help - (Apr/12/2011 )
I have a protein which is 480 a.a. (~500 kDa) in size. Can anyone recommend me a gel percentage?
I am assuming in the range of 5-10, but since this is my first time attempting this, I thought it would be great to get advice from someone who has experience.
I usually deal with a 150 kDa protein and running time and transfer time is something I have experience with. Will the larger protein have to be run longer? Is there any other obstacles I will have to deal with?
If anyone has the time to share their wisdom, I would greatly appreciate it!
a 5% gel may be what you need. it depends on the size(s) of contaminating protein(s) and desired resolution.
handling a 5% gel when setting up the transfer can be tricky. be careful, support the gel well and don't rush.
you can aid transfer by adding up to 0.05% sds in the transfer buffer (with 20% methanol). but, don't expect 100% transfer.