IL4Ra HELP!

Dahui You dahui_you at yahoo.com
Sun Jan 20 15:26:19 EST 2008


Hi, folks:
Does anybody work with anti mouse IL4R-alpha?
I am currently using BD's clone (mIL4R-M1) and it is hard to detect
 the expression of this receptor on the cells.
The attached file is the titration study I did recently. Those are
 mouse lung homogenates and stained by different dilutions of IL4Ra.
As you can tell from the figure, there isn't any distinct positive
 population therefore I gated the positive cells solely based on the isotype control (0ug
of
 the antibody on the figure). 
Sorry if this sounds stupid. But I found it is very hard to interpret
 the titration results and I need help on the following questions.
1) Is the gating correct? These cells were only stained by IL4Ra and
 the first gating was lymphocytes based on FSC and SSC scatter.
2) When it goes to 2ug and 1ug, there is a shift (to the right of x
 axis) of the negative population. What does that mean? is that non-specific bining?
3) Unfortunately, I didn't do an isotype titration. The 0ug sample
 has 0.125ug of isotype (which is recommended by the company). Should I titrate isotype
along
 with the real antibody?
4) Will 0.25ug be the best concentration to use if the experiment
 make any sense? 
5) I have so few cells considered positive, is it statistically
 meaningful?
Sorry for so many questions. I am so perplex. Any suggestions are
 highly appreciated.
Thank you in advance. 
Dahui You
Department of Biological Sciences
Louisiana State University
Baton Rouge, LA 70803
Tel:225-578-5960
Fax:225-578-2594




     
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