receptor internalisation analysis
julie.bertout at ibl.fr
Fri Sep 8 07:56:00 EDT 2006
I have a researcher interested in quantitating receptor internalisation
after different treatments.
Here is the way she used to do it :
- label receptors of interest with primary antibody and secondary antibody
- do the treatment she wants to test
- remove label from non-internalised receptor with a slightly acid
treatment (so only internalised receptors will still be fluorescent)
- analyse her samples fluorescence to see a difference.
the problem is that, as antibody/receptor afinity is different from one
receptor to another, she can't do her positive and negative controls...
for example, the receptor which she used as control (which
internalisation shouldn't change with/without treatment) has such a high
affinity with its antibody that she can't remove it with the acid treatment.
So I would like to know if there is any way to quench fluorescence for
the receptor that are not internalised or if anybody has a protocol to
quantitate internalised receptor?
I thought of comparing non-permeabilised vs permeabilised samples but
PFA fixation permeabilise cells a little, doesn't it?
Thank you for your answers,
Institut Pasteur de Lille
1 rue du professeur Calmette
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