Does anyone know of a high-molecular weight reference protein? Preferably, it has to be larger than 250kDa, but larger than 120kDa will also do. Also, it needs to be expressed in epithelial cell lines. Can I use a coomassie stain (post transfer to membrane) as an alternative for loading control, even though there are only a few bands visible on the gel (all proteins smaller than 120kDa have run off the gel)? Ponceau-S stain is not an option, since it doesn't stain well.
Dear Tanja,
You might try looking at cytoskeletal proteins. I have seen vinculin (116 kDa) reported as a loading control, and I have used spectrin (240 kDa) as a loading control in Drosophila westerns.
I've also explored the use of topoisomerase as a loading control - topoisomerase IIβ (180 kDa) protein levels are reportedly fairly stable throughout the cell cycle.
As for the membrane stain, unless you are using IR secondary antibodies for detection, amido black is a far more sensitive alternative to Ponceau S, and has the bonus of fixing proteins to the membrane since it staining and destaining involves the use of methanol. Spyro Ruby Red is an expensive alternative to Ponceau S that's compatible with IR secondaries.
I suggest to you the HMW standards kits of Biorad or Phamacia (it belongs now to GE Healthcare).
For example, the HMW kit of GE is composed of: Myosin: 25 µg, 220000 (Mr), Macroglobulin: 100 µg, 170000 (Mr), b-Galactosidase: 16 µg, 116000 (Mr), Transferrin: 17 µg, 76000 (Mr), Glutamate dehydrogenase: 18 µg, 53000 (Mr).
http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/17061501
Chockry, I believe that Tanja requires a loading control found in her cell lysate rather than these standards which are useful for sizing rather than loading controls.
Good to know that this product exists though.
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