I have transfected melanoma cells with lentivirus. The knock down worked really well, but the scramble cells show an increase in the expression of my target protein on western blot. I have also done other experiments with the cells and the scramble is just completelly different from the wild type.
I don't feel comfortable comparing the knock down with the scramble because I think they are just a completely different cell that don't relate to the wild type.
Selection antibiotic is Puromycin.
Can any one please give me any suggestion of why this might be happening or some literature I can use to explain this?
Thank you in advance,
Your scramble transduced cells (transduction for viral vector: transfection is for plasmids etc) have been infected with a virus: this will inevitably induce transcriptional programs that differ from non-transduced cells, even if the construct used in the vector is scrambled. They have also been subjected to puromycin selection, another variation from non-transduced.
This is the whole purpose of doing a scramble control: simply comparing knockdown with non-transduced cells introduces a potentially huge variable (virus infection) that you're not controlling for.
It may simply be that your GOI is virally responsive.
One way to confirm this is to prepare several scramble constructs and see if all of them induce the same upregulation.
How are you normalising your westerns, incidentally? How certain are you your loading control is not virally-modified?
Have you checked whether you scramble plasmid sequence?
Also, I think sometime WB is unstable, sometime even the same sample it needs to run many time to get stable result. Maybe you can repeat your WB experiments.
Hi, Bethania Piva.
Everytime we have lots of different things working on an experiment, variations arise.
I do agree with John Hildyard and I guess you could do some experiments (If possible to check it and give you more reliable ideas and results) ...
Your could do the other scrambled constructs and check it, the transfection vector it self alone and even treat your cells with puromycin and check them for the protein experssion by WB. Maybe, then, you can find out a way to normalize it all.
Still in time, maybe you could normalize it by your target protein comapring it to total protein expression of your cells.
All the best.
Hi,
To all thank you for all the answers and sorry for the delayed response. I will try the suggested approaches :)
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