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Problem with pErk staining and signal intensity?

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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Wed Nov 26, 2014 9:09 am

Problem with pErk staining and signal intensity?

FDCP1 cells were stimulated with IL3 and stained for pErk Yb171, pStat5 5Nd150 and pAkt Sm152.
The basal level of pErk appears to be not stained properly and very less cells are possitive to pErk and signal for stimulated pErk is too high. I am sure that i have added same amount of antibody and same amount of cells in both tubes. here is the screen shots. I am thinking what would be the reason ? .
Attachments
Screen Shot 2014-11-26 at 9.58.51 AM.png
Contour map
Screen Shot 2014-11-26 at 9.56.56 AM.png
Histogram overlay
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ianfrank

Contributor

Posts: 29

Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Wed Jan 14, 2015 6:27 pm

Re: Problem with pErk staining and signal intensity?

I'm sorry I don't have any experience with the pERK staining. Your pSTAT5-150 staining looks pretty good. I haven't been able to get this to work consistently. Have you been able to get consistent results with the pSTAT5?

Ian
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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Thu Jan 15, 2015 7:01 am

Re: Problem with pErk staining and signal intensity?

Hi Frank,

yes if u stimulate cells with IL-3 it strongly activates pStat5. for pStat5 i always get consistent results both in CyTOF and Flowcytometry. It depends on what type of cells and cytokines.

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