Analyzing outer mitochondrial membrane proteins via WB - (Apr/08/2010 )

Hi all;

I have two questions on analyzing mitochondrial outer membrane proteins. I have two proteins, one is a beta barrel intermembrane protein and one is C-terminally anchored in the outer mitochondrial membrane. The beta barrel has a c-myc tag in a cytoplasmic loop and the c-terminal anchored one has a n-terminal 6XHis tag. I am working with these both in yeast and EpH4 mammalian cells

I would like to confirm the localization of these proteins to the mitochondrial outer membrane. Both colocalize with mitochondria in vivo in EpH4 mammalian cells as can seen via immunofluorescence. However, I can't get them to show up on a western blot. I have checked my antibodies with bacterial lysate controls and they are all working fine.

After transfection, I pellet my mammalian cells and incubate for 30 min on ice in 1XRIPA buffer with frequent vortexing. I then add 2X laemli loading buffer and incubate on ice for another 5-10 min with frequent vortexing. I do not heat the sample as I have read that heat can cause membrane proteins to aggregate. I then load the sample onto and SDS-PAGE gel. Yes my protein is making it onto the membrane and no I am not overloading the sample (As checked by coommassie staining and ponceou S)

But i get no bands on my WB, just some slight background.

On the yeast side, the proteins are induced via growth in galactose and I use isolated mitochondria samples as well as spheroplasted whole yeast. I basically follow the same procedure as detailed above.

I don't know what else to do. My best guess is that the proteins are still stuck in an insoluble membrane part, but the RIPA buffer has TritonX-100, SDS and sodium deoxycholate so I figured the membranes should be pretty well dissolved. Please, i need some advice! Can anyone help me?

Cheers!
Kadiya3

I take it you are using protease inhibitors during lysis?

Have you tried doing a mitochondrial fractionation?

bob1 on Apr 8 2010, 05:30 PM said:

membrane proteins can be very tricky due to the hydrophobic regions in the protein. have you tried to detect a mitochondrial membrane marker protein like VDAC1?
Maybe its better to do a membrane protein extraction instead of a total extract (for instance, I cannot detect my membrane protein in total extracts, just in the membrane preparations)

tea-test on Apr 9 2010, 05:11 AM said:

hi, i'm using the membrane protein extraction kit from fermentas. you get high yield of membrane protein but if the cells are detaching very easily maybe this kit is not so good. they are shipping free samples for three preparations.

Have you checked if your myc and his-antibodies work on other tagged proteins?

tea-test on Apr 9 2010, 08:47 AM said: