Do you isolate the membranes before biotinylation? Can you concentrate the elutions, ie with small ultra amicon spin concentrators? Or do buffer exchange with a small spin column to get rid of the DTT? Do the proteins need to be active.

Coomasie plus from Pierce will work for the protein assay. Maybe test a buffer control.

If you are isolating membrane proteins, it may be best to partially purify the membranes first from the rest of the brain. I would think you are doing this already. If not, I have a protocol.

If the protein does not need to be active, you can elute with acid from the biotin column, and TCA precipitate to concentrate if necessary.

Hi Marcia,

Thank you very much for your reply and help it is very helpful, I am rather naive in WB and biotinylation, so please excuse any basic question form my side.

We do not care about activity of protein. I am interested in quantification of the protein before and after treatment.

As you mentioned, I have not done membrane isolation as well as purification prior to biotinylate it. We dont want any cytoplasmic contamination. Because our protein is present in cytoplasm in endosomes. But still it will be helpful if you could give protocol for the same.

I checked Coomasie plus from Pierce is a very good suggestion, I will use it.

If you want to isolate membranes, here is a good protocol.

Homogenize brain in , 50 mM HEPES or TRIS, pH 8, 200mM NaCl, plus protease inhibitor cocktail pills from Roche diagnostics or sigma cocktail with EDTA. Spin at 2000 xg and discard debris.

Spin at 100000 x g. Then take pellet and resuspend in 0.25M sucrose, 50 mM HEPES or TRIS, pH8, and spin 100000 x g to pellet membranes. Wash by resuspending in sucrose buffer again, and pellet again. Then for biotinylation, resuspend the membranes in whatever biotinylation buffer you use. Do the biotinylation, then add The sucrose/ buffer and spin at 100,000 xg again to wash away any non membrane proteins. Do this twice. Once you have the washed pellet, you can resuspend in a small volume, and add right to SDS/ loading dye buffer for the Western. If need be, solubilize the pellet first in RIPA buffer, and then do the protein assay. Then put in loading dye and run the gel.

If you want to use your procedure, ie maybe before membrane isolaTIon, which is probably better, then TCAprecipitation or ultra amicon spin concentration after biotinylation can be used to concentrate the protein before loading. And I would try to use strptavidin with an acid wash. The protocol for this can be found probably in the Pierce catalog or on the internet.

Thank you very much Marcia. we do not want any contamination from endosome or any other intracellular organelle since our protein is likely to be present in intracellular membrane (but in inactive form - that we need to exclude from our quantification). So we are not been able to isolate membrane fraction using ultracentrifuge and run the WB. Hence we decided to first tag all membrane expressed proteins using biotinylation and subsequently purify biotin tagged proteins using streptavidin beads (cell signaling - #3419) in addition to that we also tried Pierce Cell Surface Protein Isolation Kit, unfortunately neither of them worked for us.

I guess now you will get complete picture of our problem and hopefully will have some possible suggestions.

Thanks indeed

Can you make brain slices and try staining with an ab to your protein?

thank you Marcia, we do not want to do immuno-histochemistry, I hope biotinylation will work... finger crossed :)

Hi Marcia

I read with interest your answer about membrane-protein fraction isolation in brain. I'm trying to do so myself and i would like to ask you two questions:

- First one is if that protocol isolates all membrane fraction or just myelin. I used a protocol for myelin isolation that is rather similar. Now i want to isolate all membrane fractions, regardless of them being neuronal or glial and yours seems easy to do

- In case it is useful for both myelin and neuron membrane proteins, do you have that protocol written or do you of any reference that details it? Mostly to know the centrifugation times, exact procedures and so on.

Thank you very much

Hi Tushar,

I am trying to do the same as you. Isolate only CELL SURFACE proteins (avoid contamination from any cytoplasmic material including organelle membranes)  in rat hippocampal tissue. Did you figure out how to do it? I saw a JOVE article but before I start, wanted to check other protocols. Any thoughts?

Thanks

Nisha

http://www.jove.com/video/51240/brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Hi Tushar and Nissha (or others),

 Did either of you (or others) find a good protocol to isolate the cell surface proteins from brain slice tissue? If so, I'd very much appreciate it if you would contact me an share a good protocol for doing this.Thanks so much  -Andrew

Hi all, I'm a bit late on this thread but I too have been trying this technique on a potassium channel in rat neurons from brain slices. I have so far had partial success and perhaps some of my experience would be helpful to others. First, I found magnetic beads much easier to work with than non-magnetic. Instead of spinning down you can just use a cheap rare-earth magnet to pull the beads quickly away from the bottom of the vial you are working in. Second, I was successful verifying slice biotinylation by using streptavidin-fluorophore conjugate to probe for biotin on a test blot of my lysates. (I'm using a fluorescent scanner for the blot imaging).  Run some lanes of normal lysates and some from biotinylated slices of the same critter. They should look dramatically different. There will be some native biotin in the non-biotinylated lysate, but not much compared to your biotinylated lysates. Make sure that is working first. Finally, I was able to try different ratios of bead-slurry to input protein to figure out the optimal balance of beads to protein concentration without using too much lysate yet getting a strong signal off the beads. Using a sodium-potassium-ATPase antibody, or other robust signal, I can see the pull down is working for a membrane protein. However, alas, I am still trying to get enough signal for my potassium channel of interest as there are no good antibodies available for that (yet) :(  Hope any of these ideas are useful to others. The jove video got me started but you need to check your steps yourself and test for optimal ratios etc. Get the absolute best antibody signal to total-lysate first, because what comes off the beads will be much weaker. (Apparently much of membrane proteins are in the ER and Golgi and transport vesicles).