[Cytometry] LaminB as a loading control for western blot, why not work for flow cytometry?

Dynlacht, Joseph R jdynlach at iupui.edu
Tue Feb 11 12:17:47 EST 2014


Hi Shirley,

I can't tell you why you are seeing things with flow that you are not
seeing with Western blotting, but I'm not surprised that you might see a
difference in lamin B in treated vs. untreated samples.  Lamin B content
can potentially change if the treatment induces a stress response.  We
observed many years ago that lamin B increases after heat shock, much like
a heat shock protein.  Flow is also very good at picking up the
degradation of lamin B during cell death;  I can't remember our
experiments (it's been about 15 years), but I seem to remember that we
would sometimes see degradation with flow slightly sooner than we would
see it with WB.  Bottom line is that I would not recommend using lamin B
as a loading control unless you are definitely sure that you are not
inducing a stress response, cell cycle perturbations, or cell death during
the time of observation.

Hope this helps.

Joe

Joseph Dynlacht, Ph. D.
Associate Professor
Department of Radiation Oncology
Indiana University School of Medicine
Indiana Cancer Pavilion, RT041
Indianapolis, IN 46202
Phone:  (317) 278-3882
FAX:  (317) 278-0405
E-mail:  jdynlach at iupui.edu






On 2/10/14 7:06 PM, "Shirley Shen" <shangs at unlv.nevada.edu> wrote:

>Hi All,
>
>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...)
>
>Thanks,
>
>
>-- 
>Shirley Shen
>Genomics Facility; SEB, Rm 3124,
>University of Nevada Las Vegas
>4505 Maryland PKWY
>Las Vegas, NV 89154-4022
>
>702-895-1057




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