I am biotinylating with Sulfo-NHS-LC-Biotin, dissolved in PBS (1 mg / mL) changed to a pH of 8. I added biotin for 30 minutes on ice to a layer of endothelial cells plated on a collagen matrix. I then washed 6X and added Streptavidin conjugated to Alexa488 (conjugated in house) at RT for 1 hour, washed 3x, fixed with 2% PFA, and then washed and visualized. You get a lot of nonspecific fluorescence coming from the collagen matrix, but none of the cells are stained. Anything I might be doing wrong?
Hi Margarette,
it looks like the antibodies ( or maybe also biotin- not sure about this protocol) are not getting in the cell. Typically for IF you have to perm cells with either detergent or fixatives like methanol that will create holes in the cell. Hope this helps,
Ana
Hi Margarette,
you need to first get rid of the collagen. Collagen is a protein as well containing several primary amines. So your Sulfo-NHS-LC-Biotin reacts with your cells but also with the collagen in your matrix. Hence, you have a homogeneous signal from your cells and matrix.
Hope that helped,
Filip
As Filip says you need to get rid of collagen if possible. If not you need to block the amino groups of collagen before plated the cells.
I am using a pH of 8, so the lysines should be the reactive group, not the amines (I believe). But, I looked it up and lysines are 3% amino acid composition of collagen. However, I cannot get rid of the collagen due to the nature of our system (transmigration assay, then labeling surface non-transmigrated cells in order to separate them out when I run FACS)....not sure if there is a way to resolve the issue without changing systems.
But keep in mind that sulfo-NHS also blocks the proteins on the surface of your cells. So block the collagen before addition of the cells. Or you could block both, the cell's proteins and the collagen, but then you need to wait until the cells produced new surface proteins before adding Sulfo-NHS-LC-Biotin.
Nevertheless, both treatments might be detrimental to your cells viability. Why don't you use a fluorescent lipophilic tracer, which incorporates into the cellmembrane?
Could you keep us updated if a protocol works for you? I'm quite interested.
Filip -
So, our treatment with just SA, versus with Biotin and SA, look exactly the same. However, this is not the case when I treat cells plated without the collagen matrix in the same manner. Thus, it looks like the collagen matrix competes with the cells simply by trapping the streptavidin, and it is thus my worry that it would do so with any treatment. However, the lipophilic tracer is something we had tossed around and may still try. Will get back to you, thanks!
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