[Cytometry] LaminB as a loading control for western blot, why not work for flow cytometry?
shangs at unlv.nevada.edu
Mon Feb 10 19:06:53 EST 2014
I have a flow client, who tried to measure p53 in nuclear samples. Each
sample was divided into two, one prepared for western blot (WB) and the
other prepared for flow. He had laminB as a loading control for WB and the
data came out as expected, the laminBs remained constant in all the
untreated and treated (with beryllium). And then he had p53 plus laminB
measured by flow in the flow samples. Lamin B constantly increased in all
the treated samples as compared to the untreated samples by the flow runs.
He also did the same thing measuring p53 along with beta-actin to whole
cells. The actin remained the same in the samples with WB but increased in
the samples with flow. For his flow runs, I had all controls set (negative
and positive, single color controls). The primary Abs p53 and laminB and
the 2nd conjugated Abs used for WB and flow are the same.
Lets just put it away what those loading controls mean to WB for seconds.
The flow client kept asking me why laminB did not remain the same with the
flow runs. Even though instinctively I did not think that it was necessary
to set the laminB as a control for the flow nuclear samples, I did not have
a clear answer to him.
WB measures amount of proteins which is relevant to the copies per cell. If
in 10 cells, 5 cells of them with 3 copies of the protein per cell (5 x 3 =
15), or only 1 cell with the protein but has 15 copies, Theoretically it
will be the same by using WB (15 vs 15), but it is significantly different
by using flow (50% vs 10%).
Questions here are,
1. why lamin B remains constant with WB but not with flow?
2. regarding to the lamin B issue above, is it because of the differences
btwn WB and flow, or something that I (or the flow client) have to be
concerned (flow sample prep, flow setting, non-specific binding...)
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