I am a total newbie in performing SDS-PAGE and I'll be happy if you could help me on this small problem and yet so troublesome for me! I am trying to see/measure collagen from cell medium and cell monolayers (fibroblasts) by SDS-PAGE.
I get no band with my collagen control with comassie stain. collagen bovine I (tendon achille) from sigma C6897 was dissolved in 0.5M acetic acid with 1mg/ml pepsin for 4 days. I tried to load the collagen solution as it is in the gel, neutralize it or even precipitate it in acetone and redilute in SDS buffer, or PBS. but still, i got no band at all. what could be the issue? collagen no solubilized enough? i am sure I put enough collagen (around 200 ug! instead of 30 ug).
what about collagen extraction from medium and cell layer? do you advise me to use pepsin to digest all proteins except collagen? sonication? triton-X?
i've tried already many different ways and still got new results. I will really appreciate if someone could enlighten me!
thank you for any advise
Protocol I used:
1) Medium samples: precipitate in acetone overnight at 4°C, centrifuge at 4,000 rpm for 10min, resuspend pellet in either SDS buffer or PBS
2) Cell layer samples: incubate cell layer at 4°C for 10 min with 1% Triton-X with protease inhibitor, precipitate in acetone, centrifuge at 4,000 rpm for 10min, resuspend pellet in either SDS buffer or PBS. Or scrap cells in a solution containing protease inhibitor, sonicate and precipitate in acetone, centrifuge at 4,000 rpm for 10min, resuspend pellet in either SDS buffer or PBS
Edited by ebnguyen, 30 May 2012 - 03:42 AM.