Immunofluorescence for Insulin Receptor - (Feb/02/2012 )
I permeabilized my cells with Triton for doing immunofluorescence studies for insulin receptor beta (C-19) antibody. If I introduce insulin to these permeabilized cells, I see nice bright fluorescence, confirming presence of insulin receptors. IF I do not add insulin, do you think I will still see fluorescence?
that condition (without insulin) should have been your control.
Yes that condition is my control cell. The question is should it produce fluorescence like the insulin treated cells or not.
you may see some, although you may expect it to be at a lower intensity if it does show.
if it is at equal (or greater) intensity then the result is independent of insulin binding or you have enough endogenous insulin to fill all of the receptors.
So the reason behind this would be that insulin IS required for the activation of the receptors and hence the brighter fluorescence? If insulin is absent there should be minimal or no fluorescence. What if these cells were NOT permeabilized? Would that bring about any changes in the effect?
no. i'm saying it could be required. that is what your experiment is to determine.
as for permeabilization, insulin should be able to get to the receptors with or without permeabilizing.
Thanks! YOu have been of a lot of help to me
You have mentioned above that for my control cells, I should see lower intensity of fluorescence. But why?
i'm assuming that the fluorescent tag on the receptor is quenched pending a conformational change caused by binding the protein (insulin in your case). it could be that binding the insulin could cause a reduction in fluorescence but then you would not be able to tell if there was binding or if there was no label on the receptor.
if there were no difference then it would not be an indicator of anything except the presence of the receptor.