I have transfected Raji B cells, an EBV infected Burkitt Lymphoma cell line, with my protein of interest expressed in an pCMV6-myc-DDK vector. At 24, 48 and 72 hours after transfection I have seen high overexpression at mRNA level compared to untransfected control, but I am not able to see overexpression of the protein on wb, only the endogenous one. In the wb I have tried both antibodies against the myc and DDK tag and against the protein itself. I have also confirmed that the transfection has been successful. On the other hand, I am able to see overexpression at protein level, doing the exact same procedure, in Jurkat T cell (meaning that there are no problems with the vector itself). My question is: does anyone know if it is the EBV can cause protein degradation/instability, or if it can somehow inhibit translation into protein due to the pCMV6 vector? Is there anything I can do to prevent this?
Thank you
Firstly, I would like to know. What is the Idea of doing transfection in EBV infected cell line as there are other cell lines like HeLa & HEK293 cells available.
Is there any specific reason for that ?
According to what I knew is EBV virus alters changes in the infected cells at transcriptome & proteome level. I shall confirm you soon whether EBV causes protein degradation.
Hi and thank you Vishnu! The only reason for using these cells are that they are the only B cell line that we have established in our lab. I understand that they are not the most ideal cells to use because they are EBV infected, but it would be nice to study overexpression in them anyway :)
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