Hi, I want to quantify the amount of collagen(s) that got secreted outside of cells to ECM through Western Blotting the media, however I'm not sure which housekeeping gene I can use in order to normalize my data. Any ideas?
Hi Denis,
To do so, we usually stain the PVDF/Nitrocellulose membrane post-transfer and before blocking with Ponceau Red solution. This is quite convenient as you can just wash it with TBS-T. After that, you can follow your normal WB procedure (blocking, washing, 1ary AB incubation, ...).
Some people are also using a highly diluted coomassie/brilliant blue staining solution the same way, but I'm not recommending it as it will be hard to completly remove it using TBS-T.
Hope this was helpful and good luck!
Alexandre Aubert I don't quite get it. How does staining with Ponceau Red solution help's me in normalizing my data?
Ponceau Red can be use to non-specifically stain proteins on PVDF/Nitrocellulose membranes. Technically, if the collagens you are looking for are the only proteins differentially produced/secreted by you cells, intensity of the Ponceau Red staining should be the same between your conditions as the total amont of protein will be similar. If this is the case, you can then compare the amont of collagens secreted (Western-blot intensity) between your different conditions. If the Ponceau Red intensity differs between conditions, this can be due to (i) a difference in cell number, (ii) an increased cell death in your condition(s), a (iii) problem in protein precipitation, or a (iv) modified (impaired or increased) protein production/secretion in your condition(s). In parallel to the collect of medium to study secreted proteins, I recommend you to extract intracellular proteins (RIPA buffer extraction) just to be safe.
- Badges
- Science method