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Immunofluorescence in HEK cells issues


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#1 Alex-Cambridge

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Posted 01 June 2010 - 06:49 AM

Hello,

I am trying to do an immunofluorescence, using a protein (RPA : Replication protein A, a single strand protein located in the nucleus). A construction has been made, using a V5-Tag to be able to make a difference between endogenous RPA and exogenous one. HEK cells have been transfected using that construction.

I am using empty vector transfected HEK cells, and untransfected HEK cells as negative controls.

As a result, i have pictures that are showing signal against V5-RPA in untransfected cells and empty vector.

I thought to possibilities :

- Dilution of the antibody is not correct. I used 1/500, perhaps do I have to dilute more.. (signal in untransfected cells has quite the same intensity in transfected cells). So maybe unspecific signal.. But the unspecific signal seems to be located in the nucleus, and not in cytoplasm that i do not understand. I suppose that if it was just a dilution matter, I should see an homogenized unspecific signal in the entire cell and not only in nucleus..?

or

- Could the antibody recognize something else in the nucleus of HEK cells that could have a similar sequence than V5-TAG (GKIPIPNPLLGLDST). I tried a BLAST, and the result is about an extracellular matrix protein...

What do you think about this ? Did someone had this problem using a V5 tag in HEK cells ?


PS: i am sorry if my english is not correct yet, I am a french master student.

Thanks for your help

#2 laurequillo

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Posted 02 June 2010 - 05:59 AM

Hello,

I am trying to do an immunofluorescence, using a protein (RPA : Replication protein A, a single strand protein located in the nucleus). A construction has been made, using a V5-Tag to be able to make a difference between endogenous RPA and exogenous one. HEK cells have been transfected using that construction.

I am using empty vector transfected HEK cells, and untransfected HEK cells as negative controls.

As a result, i have pictures that are showing signal against V5-RPA in untransfected cells and empty vector.

I thought to possibilities :

- Dilution of the antibody is not correct. I used 1/500, perhaps do I have to dilute more.. (signal in untransfected cells has quite the same intensity in transfected cells). So maybe unspecific signal.. But the unspecific signal seems to be located in the nucleus, and not in cytoplasm that i do not understand. I suppose that if it was just a dilution matter, I should see an homogenized unspecific signal in the entire cell and not only in nucleus..?

or

- Could the antibody recognize something else in the nucleus of HEK cells that could have a similar sequence than V5-TAG (GKIPIPNPLLGLDST). I tried a BLAST, and the result is about an extracellular matrix protein...

What do you think about this ? Did someone had this problem using a V5 tag in HEK cells ?


PS: i am sorry if my english is not correct yet, I am a french master student.

Thanks for your help


I would try different antibodies (primary and secondary) concentrations. Sometimes it depends of the antibody and the company. You have to be sure that you have the right ab for IF.
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