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Protein amount to load for publication figure?


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4 replies to this topic

#1 HOYAJM

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Posted 10 May 2013 - 08:56 AM

I need a figure of a SDS-PAGE gel with a couple different purified proteins. I tend to overload gels because I am just checking quality and size and just load "a few microliters". How much (in ug) protein should be loaded per well? Each sample is pure (only 1 protein) and I am looking to get a normal size band that is more intense than the ladder, but not overwhelming.

Thanks

JM

#2 Missle

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Posted 10 May 2013 - 10:08 AM

For standard Coomassie staining, I generally load about 2ug of a singular purified protein per well. You should get a crisp dark band. In my experience, if you go much over 5ug your band will start to become more blob and less band.

#3 mdfenko

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Posted 10 May 2013 - 10:10 AM

how are you going to stain the gel?

if you use coomassie then you want 1-5ug (depends on the size of the protein, width of the well, thickness of the gel, etc).

if you stain with silver then no more than ~100ng.
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#4 Tom M

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Posted 10 May 2013 - 11:20 AM

If you have enough of your purified protein, can't you just run several lanes each with a different amount of protein and then pick the amount that gives you the best image?

#5 HOYAJM

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Posted 11 May 2013 - 07:02 PM

Thanks for the suggestions. I will be coomassie staining on a 1mm gel. The sizes are 20 & 70 KDa. I will try the 2ug and see how that goes




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