I am working with BN PAGE for the first time and I am not sure about the amounts of detergent (digitonin) that should be added. Also, I am looking for methods to find the concentration of the isolated mitochondrial sample.
Because you are doing a native gel, so you want your protein to keep as much as possible its conformation, so for membrane proteins you need to add some detergent.
The concentration depends on the type of the protein, but since you are working with digitonin, you may keep 0,1% or any concentration you have used to purify your protein (if you have).
What do you mean to find the concentration? Protein concentration? You may want to use some assays, BCA etc.
The detergents allow the hydrophobic interactions to be disrupted, therefore the mitochondrial protein complex, which is a transmembrane protein complex, is released from the mt membrane.
Thank you Jorgaq and Vipul for the answers. Also, the BioRad powerpack gives an error message after the overnight run and the run stops in the middle in cold room. Any suggestions for a successful gel run?
What %age of Acrylamide are you using? What are the power settings that you are using ?
I am using 4% and 10 % gels. I maintained a constant volatge and less than 15 mA current for overnight (35 V)
BN-PAGE of mitochondrial proteins has been described in doi:10.1006/abio.1994.1112.
It is a native PAGE.
Thank you Engelbert Buxbaum for the link
The size that I am looking for is 17 kDa and I was using 100 V
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