We are culturing different types of primary stroma and we want to use the supernatants in some experiments. But how can we equalize the supernatants? For westernblotting you can use b-actin, is there a stable factor in supernatant that we can use to compare to? All ideas and suggestions are welcome!
If you are using serum free medium, we normally ponceau stain our membrane and image it before blocking and use the bands to show equal and stable loading. There are no good loadings controls known for secretory proteins. Some people use BSA however BSA/albumin is not a great loading control as it will normalize for the amounts loaded but not for number of cells.
Thanks, I will keep that in mind. How much supernatant do you need for running a blot and stain with ponceau? How works the BSA loading control, because the number of cells is not that important for me, the amount of protein is the most important for us...
Usually around 60-70ug quantified by bradford or BCA, that would be around 40uL, it depends. The BSA loading control works as a standard westerblot using anti BSA antibodies, there are several comercial ones with very good results.
- Badges
- Science topic