Hi Kyle

We use cells on collagen coated surfaces as well. Yes, they do contribute to a good deal of the total protein signal on chromogenic methods and turn up significantly on WB, which can interfere with immunodetection of your protein of interest in the 50-60 kDa range.

One way of controlling is to have a few wells with collagen without cells. In addition to the standards used in protein assays, this can help provide a better normalization of the cells+collagen samples.

Hope this helps

Aparajita

Hello Aparajita,

Thanks so much for your response! That makes sense, I'll consider that once we are ready to begin culturing our cells. Does this help with all assays or just total protein assays (for WB, are collagen and collagen+cell bands discernable from each other)?

In my experience, type I collagen is challenging to detect via Western for many cell lines. I recommend Southern Biotech’s antibody for detection and using a positive control lane.

I’m not sure if you can differentiate between cell and substrate type I collagen with immunological methods.

Another consideration is collagenase/TIMP activities of your cells-due to degradation, by the time you terminate your experiment, you can have lost some of your substrate.