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Troubleshooting Cell Surface Biotinylation

biotinylation

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#1 electromagnetic32

electromagnetic32

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Posted 19 February 2014 - 12:06 PM

Hi-

 

I'm doing an assay where I'm trying to find out if an ER protein is being trafficked to the membrane. I have treated and untreated HepG2 cells, that I then perform cell surface biotinylation (Pierce Kit) on, and analyze by Western Blot. My controls are an intracellular protein (actin) and a membrane protein (CD47).

 

My main issue is that I keep seeing actin in my membrane fraction of untreated cells. This seems to indicate that I'm biotinylating intracellular proteins in healthy, untreated cells. My first thought was that there was a problem with my quenching reaction, so I changed my protocol to include 3 washes with PBS+100mM Glycine and then a 30min incubation on ice with PBS+100mM Glycine. However, I still see actin in my membrane fraction. 

 

I also see no CD47 whatsoever.  This is slightly less worrisome as I think the antibody just has low activity, and I will probably switch to labeling Na-K-ATPase.  Hopefully that will give a better result.

 

Given these issues, I'm currently at a loss as to why I can't even get my controls to work.

 

Any tips or ideas would be much appreciated! Thanks!







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