Western blot to CD44? (View forum version)



Guada

Posted 20 September 2013 - 01:12 PM

Hello everyone!

 

I am having a terrible time trying to get the WB against the CD44 protein to work. The antibody I am using recognizes CD44, and I could make it work two times but conditions never repeat!! Sometimes it works beautifully and other times not at all, it does not see anything. I know I have cell lysates since I am using other cell protein as control and it is there. Did anybody have this or similar issue and could resolve it? Or do you have any ideas of why is this happening?

 

Thank you very much!!

 

Guada


theo22

Posted 23 September 2013 - 07:41 AM

I have had the same problem when I tried to detect CD44 by FACS. Then I found that CD44 was induced by hypoxia in my cells. Maybe this also interferes with CD44 expression in your cells.


Guada

Posted 25 September 2013 - 12:26 PM

Hi Theo,

 

Thank you very much for your answer. That could actually make sense, could I ask you which cells were you using? I am trying Hela cells at the moment.

 

Thanks again!

 

Guada


theo22

Posted 25 September 2013 - 12:58 PM

Hi Guada,

 

HeLa cells are responsive to hypoxia in the sense that they will induce (stabilize) Hif1alpha. I did my experiments with the sponteneous immortalised "normal" breast cell line MCF10a