WB: protein amount/ lane ? - What is the optimal protein amount per lane in WB ? (Jun/26/2007 )

:rolleyes:Hello,

I think I have a very simple question, but we have not done Western Blots very often at our laboratroy. We wanted to do a SDS-PAGE. Yesterday we measured the amount of proteins (Bradford). Now to our question: What amount of protein do we have to load per lane to get optimal conditions for detection ? On what does the amount of protein loaded depend (does it depend on s.th.) ?

Thanks a lot for your answers !

Maria

Hi, I think you should start with an amount of 25-30 µg protein per lane. This is what I usually use. You can also take more or less, depending on the protein you want to detect and the amount in which it is expressed in your cells. Also, the quality of you antibody can play a role for the signal strength. But just start with 30 µg for all samples and see what you get.

Good luck!

You can make a test-gel, on witch you load from 5 to 50 ug and see what concentration is suitable for your protein and your antibody. The total amount of your protein in your sample sure is one important criteria, but the strenght of your antibody is too. For example, we often blot for p-JNK. With an old antibody, we needed 50 ug to see a good band. Now, with the antibody from another company, 10 ug is enough. So i suggest you test theses condition before doing your actual experiment.

The Bearer is right, don't forget the gel size. If you run very large gels, you need masses of your protein, so start with mini-gels.

We use 80x80mm (1mm thick) gel and usually 10-30ug per well is enough for SDS-PAGE and following Western-blot (peroxidase conjugated secondary antibodies developed with chromogenic substrate - DAB).

I have obtained good bands even with 70 ug (minigels) but that´s not very usual I guess