Dear all,
My protein stays in the nucleus and in the cytoplasm of the cancer cell, but I concern only nuclear immunoreactivity. Can I detect a protein of interest buy using indirect staining for flow cytometry?
Thank you all.
hello, Trang Nguyen. may be you can try isolating the nuclei at first?
Yah, I would suggest you to isolate the nuclear fraction and then look for your protein.
Also, you can perform indirect immunoflourescence.
You could try to permeabilize only the surface membrane in one tube and both the surface and nuclear membrane in a second tube. Stain both and compare the staining intensities. But I think your best bet will be to use an imaging flow cytometer such as the Amnis. Add a nuclear dye such as Ho and use the images to help you determine and analyze nuclear staining of your protein.
Thank you all for your suggestion.
But I have to check the correlation of this protein expression to another surface protein. So I think isolate nuclear protein first is not suitable.
@ Lynda Guzik: would you kindly tell me how can we permeabilize both surface and nuclear membrane? Normally, I use FCM Permeabilization buffer 1X to permeabilize surface membrane.
This is not something that I do, so I don't have specifics. I have heard that short incubations with 0.1% saponin will only perm the surface membrane, while longer incubations with the same buffer will perm both membranes. I know that at 30min, I get perm of both membranes. I think this sort of thing may be very hard to control consistently as well as prove that one tube only shows cytoplasmic staining while the other shows both nuclear and cytoplasmic staining which is why I reccommended using the Amnis. I would talk to the tech helpline at wherever you are buying your perm buffer. They should have more information. Good luck.
So many thank for your kindly advice, Ms. Lynda.
I'm afraid that my lab's condition will not be allowed using the Amnis. So I will try to perm with longer time.
Kind Regard,
Hi Trang.
Did you try NXTRACT kit from Sigma (very good)? It will help you to isolate protein extracts from nuclei and Cytoplasm. Once you have these extracts, perform western blotting for your protein of interest with both of them.
Confocal microscopy would be very helpful as well. But only confocal would not be strong enough to prove it. My advise is to do both, if possible: The western and confocal. This would make a "strong" work.
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