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> A certain molecule is expressed on the surface of two different cell lines
> at similar levels, but (probably) in two different conformations. I have a
> monoclonal that (probably!) discriminates between the two conformations. In
> comparing the staining pattern of these two cell lines I face the
> following problem.
> The (no first Ab) conjugate controls for these two cell lines stain at two
> different levels, so that it becomes impossible to compare the test
> stainings.
> 1)If I change the voltage for one cell line to match its control staining
> to that of the other, won't it bring about a more dramatic (and unreal)
> effect on the test staining for this particular cell line?
> 2)Or can I just superimpose the two control stainings and compare then
> compare the stainings with the monoclonal in question?
> If anyone could throw some light on this aspect of FACS data analysis I
> will be really greatful.
Sanjeev,
- From the information you give, it is difficult to make very specific
suggestions. If you are trying to work out whether populations are simply
positive or negative for each sample, and they form a biphasic distribution,
then you have no problem, compare each positive staining with its own
control. If however you are looking at shifts in fluorescence intensity
within a single population in each case, I would suggest that you calculate
the ratio between the median value for labeled and control cells for each
line, and compare those. By the way, are your cell lines of different size,
because that may have an effect on both specific and non-specific
binding. This is probably important in your experiment if you are trying to
sort out the relative expression of two isoforms using a single antibody.
It is difficult to be specific with the limited information you give
Have fun
Mike Salmon
Department of Rheumatology, Birmingham University UK.
E-mail: salmonm@rheuma.bham.ac.uk
Tel: 44 (0)21 414 6780
FAX: 44 (0)21 414 6794
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