I want to be able to look at the contents of a vesicle with both immunofluorescence and western blot. Are there ways to permeablize cells to get an antibody into the vesicle but not burst the vesicle? Are there ways to isolate the vesicles themselves so I can lyse them and analyze their contents?
Hi.
Vesicles usually carry the proprieties of the cells they're from, so u could use any antibody to stain the "mother" cell. i would not suggest any permeabilization way, i guess the membrane would be broken easily.
Hi, what kind the vesicle do you mean. If it is on the body of animal you can remove it surgically without destructing, just ask a vet to assist you.
Hi,
Please specify what do you mean by "vesicle". Do you mean synaptic vesicles?
Thanks
Boaz
I agree with Boaz, if you can specify a little bit more what you are working on, it will be easier to reply....
However if you are working with cell culture, after normal fixation (para 1 to 2%) and permeabilization (saponin 1%) you should be able to stain the content of intracellular vesicles. You must be sure however if your antibody works on fixed samples and if it recognize folded or unfolded protein. For WB you should refer to a centrifugation method that allows you the separation of vesicles after cell lysis or you can consider to separate just the cytoplasmic extract and run WB. In this latter case however you will have a smaller amount of your protein(s) of interest and possibly you will be not able to obtain a clear signal for quantification.
Regards and good luck,
Maurizio
- Badges
- Science method