2 replies to this topic

[mlanewal]

My current work involves looking at certain protein levels in skin (working in mice currently).  I have been isolating the protein through the use of Pierce's T-PER reagent (http://www.piercenet...?fldID=06010425), to which I add protease and phosphatase inhibitors.  My BCA assay tells me that I have plenty of protein in the sample, but my Western is yielding faint bands at the appropriate sizes with much larger bands located at the base of the membrane.  I'm reading the gel using a LI-COR Odyssey, if that makes any difference.

My question is this: is there a better way to isolate protein from skin?  My PI and I are worried that we are destroying a good portion of the protein during the isolation.  We've talked about dounce homogenization, which would be time consuming but doable.  I've also given some thought to digesting the samples with collagenase, but I'm worried about unintended consequences from that.  Any suggestions would be appreciated.

[Inmost sun]

mlanewal, on Feb 22 2010, 04:02 PM, said:

My current work involves looking at certain protein levels in skin (working in mice currently).  I have been isolating the protein through the use of Pierce's T-PER reagent (http://www.piercenet...?fldID=06010425), to which I add protease and phosphatase inhibitors.  My BCA assay tells me that I have plenty of protein in the sample, but my Western is yielding faint bands at the appropriate sizes with much larger bands located at the base of the membrane.  I'm reading the gel using a LI-COR Odyssey, if that makes any difference.

My question is this: is there a better way to isolate protein from skin?  My PI and I are worried that we are destroying a good portion of the protein during the isolation.  We've talked about dounce homogenization, which would be time consuming but doable.  I've also given some thought to digesting the samples with collagenase, but I'm worried about unintended consequences from that.  Any suggestions would be appreciated.

you seem to have massive problems with protease activity; improve protease inhibition; homogeneize fast and with cooling;

[swanny]

Without really knowing any details, I'd start with suggesting you keep things as cold as possible. Not sure if you can snap-freeze the samples, and homogenise frozen, but it might be worth a look.

Also, from near-complete naivety, could your protein be glycosylated? That might explain the high MWt protein you are seeing. Try treating some sample with PNGase F to remove N-linked glycosylation. I believe you can similarly remove O-linked sugars.