receptor internalisation analysis

Julie Bertout julie.bertout at ibl.fr
Fri Sep 8 07:56:00 EDT 2006


Hi,

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,

Julie Bertout
cytometry lab
Institut Pasteur de Lille
1 rue du professeur Calmette
59800 Lille
France






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