Hi everyone!, I´d like to transfect cells with a GFP expression vector and then measure the amounts of some membrane proteins just in the transfected cells (GFP positive cells) by flow cytometry. My question is: what secondary antibody can I use??. I mean, if a use a PE conjugated Ab I think that probably it wouldn't be right, because the GFP and PE ranges overlap. I hope you'll understand my question.

Thanks for your help

---

There's no problem on using GFP and PE together as long as you compensate your samples properly. Just remember to have a non-stained sample (no-GFP, no-PE) to set your backgound fluorescence, a GFP only sample, and a PE only sample to compensate. That way you can be sure that if your cells glow GFP and PE is because they are double positives.
On the other hand and if you can use other stains, I'd recomend you use APC as this does not interfere with GFP.

---

Hi "almost a doctor" and thanks for your response. I think that we don´t know how to compensate correctly, so maybe we'll try to use a secondary APC conjugate antibody. I was looking for it in the Internet and I haven´t found it yet. Just another question, what about a Texas Red antibody (with GFP remeber)??.

Thanks again

---

Texas red will give you the same problems as PE. You'll probably have less red in the green, but you'll still see green in the red.
In my experience good compensation comes with practice, and a lot of trial and error. Is a case of making sure that when you have GFP only you don't see any fluorescence in the red channel (i.e.FL2), and when you have only PE (or Texas Red) you don't see any fluoresence in the green (i.e.FL1)

Can I ask you what Flow Cytometer are you using? I know with BD's LSR you can compensate after acquiring so you could always as for help later, I've only used this once though.

As I said before just make sure you have the right controls: NO staining, and SINGLE staining to make sure things look like they should.

---

Hi again, we are using a BD FAC-Scalibur.
As far as the compensation is concerned, someone told me it's so difficult to compensate if your protein has low expression levels (and that's my case), but I don't know if it's true.

Thank you again for your help...

---