SDS page of acid soluble collagen - (Apr/30/2013 )
I am using a acid soluble collagen from bovine source..I am running a 7% resolving gel to separate my protein. I am facing an issue with collagen solubility and neutralization. I have tried several procedure yet I find it difficult to get a band. Any suggestions...Thank you
By
Shankar
can you tell us more about what you tried?
have you tried denaturing with sds and not neutralizing (or neutralizing only until the bromphenol blue turns blue again)?
with me I have a bovine collagen....as I have it in a powdered form, I solubilize my collagen using 0.5 M acetic acid...then I neutralize it and heat my samples at 95 degrees for 10 mins and then I run my samples...yet I do not get a band...any possible suggestions or reasons..
I guess after you had neutralized your acetic acid, the collagen precipitated?
Maybe you try to use urea/thiourea buffer with CHAPS?
0.5M is a high concentration of acetic acid to use for solubilization. sigma recommends using water adjusted to pH 3 with acetic acid to prepare 5mg/ml collagen (will give you an opalescent, colorless solution).
worthington suggests 0.075M sodium citrate buffer, pH 4.3-4.5, to prepare 6mg/ml collagen. (both from calf skin).
you can take an aliquot from one of these, add sds loading buffer, then neutralize with tris base until the solution turns blue, boil and load onto the gel.