I try to resolve collagen on SDS-PAGE. My goal is to resolve the collagen extracted from skin scaffolds. I use ThermoFisher Bolt mini gels. I can see the protein ladder migrating and also I can see BSA positive control. Yet I can see neither the collagen type I from Sigma nor the collagen from my samples. I dissolve collagen at 8 mg/mL in 0.5 M acetic acid. I tried to desalt it and add to the LDS buffer, add it without desalting, trypsinize it (then I can see the band from trypsin in my gel), reduce it, I did it with heating and without heating the LDS-treated sample. Certainly, the protein is there and I cannot see it precipitating in LDS. I tried to visualise it with Coomassie stain and with the silver stain, never have I seen any band from the collagen. It is not visible at the start of the gel either.
Can anyone help?
Thanks a lot in advance.
Hi Hanna,
Acetic acid could be degradating collagen at the concentration you used. You could try with 0.02 N acetic acid, as shown in this paper:
Rajan N,et al. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protoc. 2006;1:2753-8.
Collagen is stable in 0.5M acetic acid in my experience but I dilute the acid before running gels because it can warp the gel. If your collagen is degraded, you will likely see a smear across the MW spectrum (unless you're loading wells soo lightly that there's not enough protein). What is your source of collagen? Sometimes gelatin is improperly labeled as collagen. Another possibility is that your collagen is not becoming linearized- if it remains in the triple helix and doesn't properly denature during sample prep, then you'll just have a large band at the inlet of your well or in the 300+ kDa range, which is out of range for a lot of ladders.
Jeffrey Brown yes, thank you. In the meantime I've learnt I need collagenase to linearize and resolve this protein on a gel.
Hanna Lewandowska
Please check your method again.
Moreover, have a look on the following link:
https://www.jbc.org/content/267/29/21211.full.pdf
I hope it'll help !
Hanna Lewandowska
Did you manage to perform the SDS-PAGE of your collagen samples after treating them with collagenase? Collaganase itself gives a lot of bands in the electrophoresis how did you solve this problem?
Hi Hanna,
It is possible that conditioning your collagen in 0.5 M acetic acid, if not degrading your protein, may at least negatively impact your migration. Is your sample turning yellowish when you adding your loading buffer (containing bromophenol blue) ? If yes then your sample is too acidic for proper migration. Normally your collagen should be totally denatured before migration by adding loading sample buffer and boiling it. This helps separates the triple helical structure of type I collagen into monomers. Type I collagen is composed of 2 alpha1 monomers and one alpha2 monomer, so your migration profile should look like the standard in figure 2 of this paper : https://www.frontiersin.org/10.3389/conf.fbioe.2016.01.01274/event_abstract.
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