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Problem in expression of protein cloned in pcDNA3


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#1 Niraj

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Posted 09 August 2010 - 10:22 AM

Hi,

I have an unusual problem. I am trying to express my protein of interest (POI) under the CMV promoter of pcDNA3. I have cloned the gene for POI under the 3HA tag of pcDNA3 in-frame. Three clones were confimred by DNA sequencing at the junction of the fusion. Then, all the clones were transfected into 293T cells along with pEYFP-N1 as control. The cells were fluorescing green after transfection but when I performed the Western blot there is nothing detectable using anti-HA antibodies. Then I went on to perform the immunofluorescence using the anti-HA antibodies but it failed again.

Do you have any idea what is going on here? What should be done to troble shoot this problem?

NB: The POI is adenoviral protein. pcDNA-3HA was created by me and it works great with other gene.

Hope to hear from you soon.

Regards,
Niraj

#2 NemomeN007

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Posted 10 August 2010 - 07:34 AM

Hi,

I have an unusual problem. I am trying to express my protein of interest (POI) under the CMV promoter of pcDNA3. I have cloned the gene for POI under the 3HA tag of pcDNA3 in-frame. Three clones were confimred by DNA sequencing at the junction of the fusion. Then, all the clones were transfected into 293T cells along with pEYFP-N1 as control. The cells were fluorescing green after transfection but when I performed the Western blot there is nothing detectable using anti-HA antibodies. Then I went on to perform the immunofluorescence using the anti-HA antibodies but it failed again.

Do you have any idea what is going on here? What should be done to troble shoot this problem?

NB: The POI is adenoviral protein. pcDNA-3HA was created by me and it works great with other gene.

Hope to hear from you soon.

Regards,
Niraj


I would double check to make sure that there is no signal peptide that is being cleaved between your HA tag and POI....




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