loss of activity during fractionation

Alison Leakey leakeya at citec.qld.gov.au
Thu Apr 17 21:17:42 EST 1997


I  was hoping for some advice to solve a problem I am having. I am
currently trying to estimate the size of a protein with biological
activity using size fractination. Following extraction (activity intact)
I separate proteins using a Sephacryl S-100 column using a phosphate
buffer with NaCl 0.15M (pH 7.0) at 4 degrees. The dilute fractions are
subsequently concentrated using Biomax 5K centrifugal membranes.
Activity is then measured. Unfortunately, activity is reduced by at
least 400X by the fractionation and/or concentration. There are visable
bands on the UV monitor readout and in silver-stained gels but no
activity. Any advice?

It worked with small activity the first time I did it, but not since,
despite the fact that I increased the starting sample by a factor of 4..

Are there any obvious things I am missing or suggestions to improve
stability ?


Alison Leakey
PhD Student
North Queensland Clinical School Laboratory
Ground Floor, Pathology Building,
Townsville General Hospital
Phone me at (077) 81 9662
FAX me at (077) 81 9649

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