In our lab we isolated the extracellular matrix from human adipose tissue and digested using 0.5M acetic acid. When I mixed the digested ECM with SDS loading dye (contains SDS, Bromophenol blue, Glycerol, beta mercaptoethanol) the protein got precipitated. I am not able to run the PAGE. What can be the reason for precipitation? How I can run the sample?
Homogenize fat with normal porcelain mortar, use PBS buffer with protease inhibitors and all the detergents below:
1% TWEEN/TRITON-X
0.5% sodium deoxycholate
0.1% SDS
I trained it before taking the human fat for isolation on animal fat (pig, chicken), please do it on any available before, to get handling of the method. You do it at RT, adding two volumes of your sample mass of PBS supplemented as above. When the fat is becoming nicely white, and the PBS pink/brownish your isolation is done (1 min).
Acetic acid is killing your proteins, thus you observed precipitation. I didn't noticed in your sample buffer Tris pH 6.8?
Please feel free to ask, since I've spent last two years doing it not always the proper way;)
Homogenize fat with normal porcelain mortar, use PBS buffer with protease inhibitors and all the detergents below:
1% TWEEN/TRITON-X
0.5% sodium deoxycholate
0.1% SDS
I trained it before taking the human fat for isolation on animal fat (pig, chicken), please do it on any available before, to get handling of the method. You do it at RT, adding two volumes of your sample mass of PBS supplemented as above. When the fat is becoming nicely white, and the PBS pink/brownish your isolation is done (1 min).
Acetic acid is killing your proteins, thus you observed precipitation. I didn't noticed in your sample buffer Tris pH 6.8?
Please feel free to ask, since I've spent last two years doing it not always the proper way;)
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