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Discrepancy between WB and Flow cytometry result

Flow cytometry Western Blot FACS WB

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5 replies to this topic

#1 Tabaluga

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Posted 08 April 2013 - 02:52 AM

Hi everyone,
I checked for expression of a (surface) protein in a cell line with flow cytometry and WB. According to flow cytometry (which is well established for this protein in my lab) 40% of the cells do express the protein. According to WB (which is also well established) there is no expression.
What could explain the discrepancy ? And which result would you trust ? Honestly, I'm at a loss, it's extremely strange...

Thanks...

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#2 GNANA

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Posted 08 April 2013 - 04:15 AM

I think its a matter of sensitivity, FCM is sensitive than western, as you say only 40% of cells express. Just to make sure, IP-down your protein and check whether it is a matter of visualization through straight western or expression.
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#3 Missle

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Posted 08 April 2013 - 06:01 AM

Is the antibody that you're using monoclonal or polyclonal? If it is monoclonal, it could have a conformational epitope. A conformational epitope would be maintained in Flow but destroyed in WB.

#4 leelee

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Posted 08 April 2013 - 06:50 AM

Are you using a live dead discriminator in your flow assay?

#5 Vinegar

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Posted 08 April 2013 - 11:31 AM

Is the antibody that you're using monoclonal or polyclonal? If it is monoclonal, it could have a conformational epitope. A conformational epitope would be maintained in Flow but destroyed in WB.


This was my thought as well. Not that uncommon for a monoclonal to work in an IP or FC but not on a WB.

#6 Tabaluga

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Posted 08 April 2013 - 01:03 PM

Thanks for your replies !


Is the antibody that you're using monoclonal or polyclonal? If it is monoclonal, it could have a conformational epitope. A conformational epitope would be maintained in Flow but destroyed in WB.


This was my thought as well. Not that uncommon for a monoclonal to work in an IP or FC but not on a WB.


But it can't be only with this particular cell line ? Both methods are well established for this type of cells / cell lines, the AB works fine in both WB and FACS. Just this one cell line making trouble.
By the way, I looked up old records, earlier there were no positive cells in either FACS or WB. Posted Image

Are you using a live dead discriminator in your flow assay?


No, I gate out debris etc. but don't have PI or something similar. But then again I ask myself why suddenly this one cell line should have 40 % more dead cells ? They looked fine in culture, too.

I think its a matter of sensitivity, FCM is sensitive than western, as you say only 40% of cells express.


That was my first thought too, but I think 40 % is quite an amount, and the MFI is not that low either. And now considering the old measurements I looked up, I don't think that explains it, as FACS was negative back then...

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 






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