Single stain control for 293 cell line - (Sep/02/2011 )
I am trying to transfect a GFP conjugated surface protein called CD47 (mouse) into the cell line 293. I want to check for the CD47 expression on the 293 cell surface. The only antibody that I could find that detects mouse and not human CD47 has only 2 colours, FITC and PE. FITC is out of question because of the attached GFP, and PE requires compensation if the cells also glows from GFP.
I need an antibody that is coloured PE and is expressed highly on 293 cells to do colour compensation. What would be a good antibody?
Why can't you use the GFP the protein is tagged with?
How about using labelled secondary antibodies?
Sorry, I shouldn't have used conjugated, it is more that it is on the same plasmid, but the 2 protein gets cleaved apart in the cell after transfection. I can see the expression of GFP, however I also need to prove CD47 is working and transported to the cell surface.
What do you mean by secondary antibodies?
The usual way to use antibodies is to have two or more, one primary and one secondary. It works like this...
Add your first (primary) antibody (say a rabbit antibody against CD47), let it bind, wash off unbound. Add a secondary antibody targeted against IgG from the species your first antibody was raised in (in the e.g it would be a goat or horse anti-rabbit-IgG) which can be labeled with a fluorophore.
This means that you can have "any" rabbit antibody and detect it with the anti-rabbit secondary. You can do the same for antibodies raised in any other species you care to name.
It also means you don't have to have each antibody you use conjugated with a fluorophore, so it is a lot less expensive, as the antibody companies can make secondary antibodies in bulk for relatively cheap prices. It even means you can get a range of different colours (anything from infrared to UV excitation) in the same species, so no cross-talk issues if you already have GFP in there.
Note - I don't do FACS so I am not sure if there is some constraint against doing it that way!
You could also just put the cells on a slide and do some immunofluorescence to see if your trasnfection has worked
In order to do color compensation, you don't need to use the 293 cells, you could use antibody capture beads which have a species specific capture antibody on the bead surface that you can stain with the CD47 PE antibody. You can use this for purposes of compensation.