Gummy mess after heating SDS-PAGE sample loading buffer - (Mar/27/2008 )
I saw something very unusual with my SDS-PAGE samples yesterday and I was hoping somebody may be able to provide some answers to explain what I saw. I am trying to detect a protein which is secreted into cell culture media by western blot. I took the medium (serum-free DMEM) from a single T175 flask 24 hours after replacing the medium and concentrated 20ml of media down to ~50ul using a 50KDa spin concentrator. I then added 3X SDS-PAGE loading buffer and heated at 95 degrees for 2 minutes. A control sample containing a known amount of protein was similarly treated. After heating, the control sample looked normal however the protein concentrate sample had "solidified" to a gummy mess. I don't know how much protein was in my concentrate and the loading buffer was rather old (~3 years). I have never seen something like this before. Does anybody have any idea why this might have happened?
Maybe the quantity of protein was too high for the volume?
A possible explanation...
Apparently, the presence of high concentrations of collagen may have given me the gummy mess. When collagen is heated to 95 degrees celsius, the triple helix of the collagen molecule denatures. Upon cooling, the protein renatures, however, each monomer renatures in a random fashion with respect to the triple helical region and forms a network among collagen monomers. This gives rise to a gummy substance. It's the same principle involved in making glue out of skin and sinew of animals. An old horse was sent to the glue factory where the collagen from the animal was boiled then cooled to make glue. I did exactly that in the lab yesterday except instead of a horse, I used the extracellular matrix secreted by my adherent cells.