It's a particular ECM protein that I can detect in the media well enough, especially if I concentrate the media samples. However, analyzing the concentration is more difficult due to the fact that I couldn't build the standard curve and therefore measure protein levels in the media using coomassie. Do you have recommendations for other ways to ensure equal loading of protein in samples?
Hi there,
If BSA is 0.1% in all of your samples then reading OD at 280nm using a 0.1% BSA sample as a reference should be OK to evaluate the non BSA protein content of each sample. BSA 0.1% (1mg/mL) has an OD at 280 nm of 0.67 (1cm path length).
Prefer using Bradford assay to determine protein concentration ECM in the medium.
Shamsher S. Kanwar I used Bradford assay but this is didn't work probably because the media was used as the diluent for BSA and samples.
With CBB-binding you determine total protein, if you want the concentration of "your" protein, you need a specific assay:
- Do you have an antibody against the protein? Then you could use ELISA or (quicker and more sensitive) spot-blots.
- Alternatively, you could do an SDS-PAGE and quantify band intensity in scanned gels with NIH-Image (less reliable, but doesn't require antibodies).
- If your protein has a measurable activity (e.g., enzyme or effect on cells in culture), you could use that for quantitation.
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