Hi all,
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.
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#2
mdfenko
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Posted 05 May 2011 - 10:21 AM
yes, you should use a refrigerated centrifuge. most enzymes are more stable when maintained at cooler temperatures during purification.
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.
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.
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#3
Eternalist
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Posted 09 May 2011 - 10:38 AM
Thanks for the input MDfenko, after some time I figured out what I was doing wrong. The procedure was right, but sampling method flawed.
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.
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.
