Need help with extracting collagen

[cem54]

Hello all!

 

I am an undergraduate working on a project where we are trying to extract collagen from the fat pad of a human (in the heel). We attempted to extract collagen from fresh tissue samples of a human heel pad using the protocol from Pacak, MacKay, and Cowan (2014, Jove). Then we attempted to electrophoresis the sample in a 7.5% SDS-PAGE gel along with a protein ladder and a sample of standard collagen from Sigma Aldrich (5 mg/mL). When we developed the gel (staining overnight and destaining overnight), all that we saw run was the ladder and the standard from Sigma Aldrich. I am very confused on what might have happened since we followed the procedure exactly and since we also ran samples of the waste that was generated during several parts of the extraction protocol. I am kind of at a loss, so any help would be appreciated. I don't really know what to do next. Thanks!

[CharonY]

Did you do protein determination determination esp. on the filtered fractions? Protein extraction is often a matter of practice, though complete loss esp. in case of something relative sturdy such as collagen is a bit unusual. Since you did not even see a smear it appears that you lost it completely rather than having degradation, which is more common.

Edited June 17, 2016 by CharonY

[cem54]

So you're saying that it's likely that I lost most/all the collagen due to a bad purification process? It's entirely possible; it was the first time doing this protocol. And you're suggesting doing something like a BCA before electrophoresis? Ok I will pass that along to my PI. Thank you!

 

Any other suggestions?

Edited June 17, 2016 by cem54

[CharonY]

Just the usual things for protein work, i.e. work quick, precise and, if needed, cold to avoid degradation and contamination. However, collagen is usually fairly stable. I would therefore keep sampling at the steps where loss may occur to see at which point you lose the protein. Assuming that the sample lane was actual clear and not, e.g. a smear.