If you are using 1Ds and looking for expression using immunological
changes, the use of cell number for normalizing can cause problems because
some of your lanes will be overloaded (blocking of regions of the blot,
obscuring of protein bands). Therefore, it is usually best to load by
total protein and be aware of the difference in cell number. If the
changes detected have that factor difference predicted from the change in
cell number, then you can look for a mechanism that ties the expression to
the cell number (like teleomere binding). Its also wise to know if the
cytoplasmic/nuclear volume in the cells is changing and to evaluate any
changes in light of those differences as well.
If you are doing 2Ds you might be able to find a large group of proteins on
a 2D gel that covary (housekeeping proteins); you can use the sum of all
those proteins to normalize one gel to another.
Katheryn Resing
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