I am doing a Western blot of an isoform of fibronectin (EDB) that is present in cancer. Normally, I would use beta-actin as a loading control. However, fibronectin is secreted into the matrix, so I have collected protein from the media. Therefore, actin will not be there. I cannot use another protein that is highly expressed in cancer, such as collagen, as a control and I am still learning about the composition of the extra-cellular matrix. So, I was planning to do some research, but I thought I would ask here too, in case anyone has done any similar work. Thanks for your help!
Cytochrome-C. It shows equal levels as far as there is no cell death in the system. Refer Supplemental figure-3 of my paper
http://www.nature.com/articles/cddiscovery20163
Hi Sarah, when you are doing western blots of cell extracts or secreted proteins the best control is to load equal amounts of protein.
Dear Sarah
Fibronectin (FN) is a ubiquitously expressed, multi-domain glycoprotein that is critical for development and that is instrumental in numerous physiologic and pathologic processes.
For more information please check the following publication: CONTROL OF FIBROBLAST FIBRONECTIN EXPRESSION AND ALTERNATIVE SPLICING VIA THE PI3K/AKT/mTOR PATHWAY. Exp Cell Res. (2010)1; 316(16): 2644–2653.
Hoping to be helpful
Marius
I will second the recommendation of determining the protein concentration of your conditioned medium to ensure loading equal amounts of protein onto your gel. Our lab also works on fibronectin and that's what we do when when looking for it in conditioned medium.
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