I want to do one protein secretion detected by Western blotting, so I harvested supernatant protein, but I don't know which protein to use as a control. We all use actin or tubulin as a control in cell lysate.
No you can not use actin or tubulin as they are not secreted. BSA/albumin is not a great loading control as it will normalize for the amounts loaded but not for number of cells. In case you are using serum free medium, we normally ponceau stain our membrane and image it before blocking and use the bands to show equal loading. There are no good housekeeping controls known for secretory proteins.
In my previous experiments when I performed western blot to assess secreted protein from cells, I used bovine serum albumin (BSA) as my control. This was possible because I performed western blot directly on the conditioned cell media the cells were cultured in. Cell culture media used was DMEM supplemented with BSA. If the cultured media you used also contain BSA chances are you can also use BSA as a control.
To get the most accurate results, it's important to use a loading control that has similar abundance to your protein of interest. If you use a highly abundant control, but your target is low abundance, the results won't be as accurate (and vice versa). The key is to be sure that BOTH the target and control are being detected within the linear dynamic range of your detection method. That range is variable and can be narrow, especially if you are using film.
Hi, Xiahui. my medium doesn't contain FBS or BSA, so BSA is good control.
No you can not use actin or tubulin as they are not secreted. BSA/albumin is not a great loading control as it will normalize for the amounts loaded but not for number of cells. In case you are using serum free medium, we normally ponceau stain our membrane and image it before blocking and use the bands to show equal loading. There are no good housekeeping controls known for secretory proteins.
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology