Active protein extract from corn
Posted 05 May 2011 - 08:19 AM
I have a protein expressed in corn (tissue) and am trying to extract and quantify the active portion. I've extracted the cellulase from tissue by homogenizing in liquid N2, folowed by bead-beating in a 50 mM NaAc solution with 0.01% trition x-100, 10 mM EDTA. Then precipitated supernatant with liquid ammonium sulfate - freezer 1 hr, centrifuge <-- does this have to be at 4 C? I don't have a refrigerated centrifuge. Resolubilized in NaAc and assayed for activity.
After back-calculating against standards I determined the protein concentration........ This was 5 months ago......I am now following the exact same protocol and cannot get an activity out of my enzyme! Does anyone have any idea what little things I may have changed that could cause this?
I also accept tips on extraction technique....as long as the protein stays active after. Thanks in advance.
Posted 05 May 2011 - 10:21 AM
how did you store the enzyme solution?
some enzymes are not stable when frozen. they are better stored at -20C in 50% glycerol.
0.5 to 2 mM dithiothreitol will maintain the reduced state of sulfhydryls in the protein.
the protein may also require a certain ionic strength for stability.
Edited by mdfenko, 05 May 2011 - 10:23 AM.
genius does what it must
i do what i get paid to do
Posted 09 May 2011 - 10:38 AM
I am not a plant-scientist and made the mistake of assuming a uniform protein concentration in different parts of the corn tissue. So when I was grinding tissue prior to extraction I was unknowingly biasing my sample through discrimination of stem and leaf material. Lesson learned.
Note: If extracting proteins from plant tissue, homogenize large batches of material for uniform sub-sampling.