I'm trying to differentiate RAW 264.7 cells into osteoclast-like cells using RANKL. By eye I can see that multi-nucleated osteoclasts are forming by day 3, but when I blot for proteins such as TRAP or Cathepsin K, I cannot detect any expression.
Anyone have any experience with western blotting for osteoclast markers in RAW cells?
Before moving on to do the western with TRAP and Cathepsin K. You can try out and do the staining for TRAP in in the differentiated cells. If you have the cells that are multinucleated osteoclasts they will pick up the TRAP stain. Then you can be sure about your blot that you are presuming are having osteoclast cells.
Hi Lesley. Its simple you need to increase the amount of protein you are loading. To get more protein you need to differentiate in a larger culture dish.
All the best.
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