I have 5 cell lines now, which has no bands on western blot if I checking my protein. To make sure about the knock-out, I PCR amplified the targeted exons and sent to sequencing (checked the bands on gel).
Unfortunately, there is no nucleotide insertion or deletion as compared to the parental cell line. How this is possible?
Any suggestions welcomed!
Hi. I think you need to check the control together when you do Western blot.
litlle mistake in western blot step can make no band(antibody or condition etc)
actually, disappearance of ptoein without DNA disruption dosen't make sense.
one of the another possiblity is off-target effect. if target gene of interest have repeated region, that could be happen.
you'd be better to check whole seqeunce of your target gene.
I hope it is helpful to you.
Hi Jaewan,
I put the gDNA of the parental cell line on the WB and I had signal there. I also develepod GAPDH to check protein level in the samples.
Did you check if your GOI/POI has a Pseudogene? Maybe you are amplifying the whole time the Pseudogene?
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