A problem with transient expression a myc-tagged protein in 293T cells - (Oct/19/2008 )
Hi, everyone,
I've got a problem with transient expression a c-myc tagged protein in 293T cells.
I cloned a gene (441bp) into a vector named pCMV-Tag3A and sequencing results was OK. The tag is in the N-terminal and no mutation at all. Both the tag and gene were in frame. But unfortunately, it just could not be detected using western blot with an antibody against c-myc. A positive plasmid was included in this experiment and it worked well and could be detected by immunoblot.
Could anyone tell me why?
How to improve?
Thank you!
-glcui-
QUOTE (glcui @ Oct 19 2008, 06:26 PM)
Hi, everyone,
I've got a problem with transient expression a c-myc tagged protein in 293T cells.
I cloned a gene (441bp) into a vector named pCMV-Tag3A and sequencing results was OK. The tag is in the N-terminal and no mutation at all. Both the tag and gene were in frame. But unfortunately, it just could not be detected using western blot with an antibody against c-myc. A positive plasmid was included in this experiment and it worked well and could be detected by immunoblot.
Could anyone tell me why?
How to improve?
Thank you!
I've got a problem with transient expression a c-myc tagged protein in 293T cells.
I cloned a gene (441bp) into a vector named pCMV-Tag3A and sequencing results was OK. The tag is in the N-terminal and no mutation at all. Both the tag and gene were in frame. But unfortunately, it just could not be detected using western blot with an antibody against c-myc. A positive plasmid was included in this experiment and it worked well and could be detected by immunoblot.
Could anyone tell me why?
How to improve?
Thank you!
Hi ya!
How old is your 293T cells? If your cells have gone through lots of passages, they become less sensitive to transfectant reagents. Ideally, for transfection, your cells should be less than 40 passages.
On the other hand, it could be your protein is toxic to the cell, and the cells were unhappy to have those proteins in them. Hence, they cleaved all of them.
Cheers.
-virus_fan-