Is there anyone out there who routinely gold-labels proteins? I have checked into this for my favorite protein (MFP), for which I alreaddy have some evidence of receptor-mediated endocytosis, I've got some nice electron micrographs of cell-surface binding at 4 degrees using a pre-fixation immunogold labeling method, but I would like to look at kinetics of trafficking via EM after switching to 37; I've tried binding the biotinylated protein to the surface, then tagging it with gold-labeled streptavidin, I've tried tagging it with gold-labelled AG-Ab complex, I've tried doing intracellular immunogold labeling after permeabilizatiojn of the cells with digitonin, but so far, none of these approaches have worked. I've done some checking into gold-labeling of the protein itself, sicne that would seem to be the simplest approach, but a) my protein has no Cys residues (which apparently makes it an unreliable substrate for the gold-labeling protocols I've looked at, b) from what I've seen, to perform gold labeling is not trivial and is best done by someone with a lot of experience of which I have none, and c) having it done commercially would be prohibitively expensive at the moment. Is there anyone out there who does this sort of thing routinely (or know someone else who does), if so, would they be willing to help me out? In return I'll make them a full collaborator (i.e. co-author) on any publications coming out of this work. This EM work is being done in parallel with multiple fluorescent colocalization of MFP with cellular markers of cellular compartments.

You may want to try some of the bi-sulfide X-linking reagents (e.g. from Pierce). The disulfides (e.g. DSS) will allow binding to the gold while the functional groups at the other end will bind to your favorite protein via amine groups. After coupling wash the beads with Tris to inactivate the unreacted groups.

evansus said:

You may want to try some of the bi-sulfide X-linking reagents (e.g. from Pierce). The disulfides (e.g. DSS) will allow binding to the gold while the functional groups at the other end will bind to your favorite protein via amine groups. After coupling wash the beads with Tris to inactivate the unreacted groups.

Thanks, I'll check these reagents out.

I am also interested in labelling heparin with gold. Does anybody know about any companies who could do it/sell it for me?

Ted said:

Is there anyone out there who routinely gold-labels proteins? I have checked into this for my favorite protein (MFP), for which I alreaddy have some evidence of receptor-mediated endocytosis, I've got some nice electron micrographs of cell-surface binding at 4 degrees using a pre-fixation immunogold labeling method, but I would like to look at kinetics of trafficking via EM after switching to 37; I've tried binding the biotinylated protein to the surface, then tagging it with gold-labeled streptavidin, I've tried tagging it with gold-labelled AG-Ab complex, I've tried doing intracellular immunogold labeling after permeabilizatiojn of the cells with digitonin, but so far, none of these approaches have worked. I've done some checking into gold-labeling of the protein itself, sicne that would seem to be the simplest approach, but a) my protein has no Cys residues (which apparently makes it an unreliable substrate for the gold-labeling protocols I've looked at, b) from what I've seen, to perform gold labeling is not trivial and is best done by someone with a lot of experience of which I have none, and c) having it done commercially would be prohibitively expensive at the moment. Is there anyone out there who does this sort of thing routinely (or know someone else who does), if so, would they be willing to help me out? In return I'll make them a full collaborator (i.e. co-author) on any publications coming out of this work. This EM work is being done in parallel with multiple fluorescent colocalization of MFP with cellular markers of cellular compartments.