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Western Blot of Large Protein Help


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#1 Biochem_newbie

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Posted 12 April 2011 - 09:21 AM

Hi there.

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!

Thank you

#2 mdfenko

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Posted 12 April 2011 - 10:30 AM

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.
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