5 replies to this topic

[Tabaluga]

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...

[GNANA]

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.

[Missle]

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.

[leelee]

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

[Vinegar]

Missle, on 08 April 2013 - 06:01 AM, said:

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.

[Tabaluga]

Thanks for your replies !

Vinegar, on 08 April 2013 - 11:31 AM, said:

Missle, on 08 April 2013 - 06:01 AM, said:

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.

leelee, on 08 April 2013 - 06:50 AM, said:

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.

GNANA, on 08 April 2013 - 04:15 AM, said:

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...